ES2416104T3 - Aldolases, nucleic acids that encode them and methods to produce and use them - Google Patents

Abstract

A method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, the derivatives of (R, R) -monatin compounds of general formula (II): ** Formula ** wherein Ra, Rb, Rc, Rd and Re each independently represent any substituent that is selected from a hydrogen atom, a hydroxyl group, a C1-alkyl group C3, a C1-C3 alkoxy group, an amino group or a halogen atom, which is selected from an iodine atom, bromine atom, chlorine atom or fluorine atom, provided that Ra, Rb, Rc, Rd, and Re are not all hydrogen and where Rb and Rc, and / or Rd and Re together form a C1-C4 alkylene group, where a pathway reaction is facilitated by a polypeptide which is selected from an isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 276 or fragments or subsequences thereof that have aldolase activity.

Description

Aldolases, nucleic acids that encode them and methods to produce and use them

Field according to the invention

The present invention relates to molecular and cellular biology and biochemistry. More specifically, the invention relates to methods for preparing polypeptides having aldolase activity, as defined in the claims.

Background of the Invention

Monatin is a high intensity sweetener that has the chemical formula:

The monatin includes two chiral centers leading to four possible stereoisomeric configurations. The R, R configuration (the "R, R stereoisomer" or "la (R, R) -monatin"); the S, S configuration (the "S, S stereoisomer" or "la (S, S) -monatin"); the R, S configuration (the "R, S stereoisomer" or "la (R, S) -monatin"); and the S, R configuration (the "S, R stereoisomer" or "la (S, R) -monatin"). As used herein, unless otherwise indicated, the term "monatin" is used to refer to compositions that include all four monatin stereoisomers, compositions that include any combination of monatin stereoisomers (such as a composition which includes only the monatin R, R and S, S stereoisomers), as well as a single isomeric form.

For the purposes of this disclosure, the carbon backbone will be numbered as illustrated above, with carbon being attached directly and covalently to the alcohol group identified as the carbon at position 2 and carbon attached directly and covalently to the amino group identified as the carbon at position 4. Accordingly, references herein an (R, R) -monatin, (S, S) -monatin, (R, S) -monatin, and (S, R) monatin mean: (2R, 4R ) -monatin, (2S, 4S) -monatin, (2R, 4S) -monatin, and (2S, 4R) -monatin, respectively, unless otherwise indicated.

It should be noted that, in the literature, the carbon backbone of monatin has also been numbered using an alternative convention, with the carbon attached to the alcohol group being carbon at position 4 and the carbon attached to the amino group being carbon at position 2. Therefore, for example, references to (2S, 4R) -monatin in this disclosure would be the same as references to (2R, 4S) -monatin in the literature using the alternative numbering convention.

Furthermore, due to the different naming conventions, monatin is known by several alternative chemical names, including: 2-hydroxy-2- (indole-3-ylmethyl) -4-aminoglutaric acid; 4-amino-2-hydroxy-2 (1H-indole-3-ylmethyl) -pentanedioic acid; 4-hydroxy-4- (3-indolylmethyl) glutamic acid and 3- (1-amino-1,3-dicarboxy-3-hydroxy-but4-yl) indole.

Certain isomeric forms of monatin can be found in the root bark of the Schlerochiton ilicifolius plant found in the Transvaal region of South Africa. United States Patent Applications Nos. 10 / 422,366 ("the '366 application") and 10 / 979,821 ("the' 821 application") disclose, among other things, polypeptides, pathways, and microorganisms for in vitro production and monatina in vivo.

Compendium

Polypeptides having aldolase activity (hereinafter "aldolases") are disclosed, including pyruvate aldolase activity such as, non-exhaustively, HMG and KHG aldolase activity, polynucleotides encoding the polypeptides, and methods for preparing and using the polypeptides and polynucleotides. In some embodiments, compositions (such as pharmaceutical compositions, fuel compositions and fuel additives, food and food additives, beverages and beverage additives, feed and feed additives, drugs and drug additives, food supplements) are also disclosed which comprise polypeptides or polynucleotides. These compositions can be formulated in a variety of forms, such as tablets, gels, pills, implants, liquids, sprays, films, micelles, powders, food, feed granules, or any type of encapsulated form.

Aldolases and / or compositions thereof can be useful in pharmaceutical, industrial and / or agricultural contexts.

Aldolases and / or compositions thereof can be useful for forming or cleaving carbon-carbon bonds.

Aldolases are provided that catalyze reactions that form carbon-carbon bonds between an alpha-ketoacid acceptor and a pyruvate or a pyruvate-derived donor (see reaction scheme below). The acceptor can also be a ketone or an aldehyde. Furthermore, aldolases are provided that have 4-hydroxy-2-oxyglutarate aldolase activity (such as 2-keto-4-hydroxyglutarate aldolase, 2-oxo-4-hydroxyglutarate aldolase, KHG-aldolase, EC 4.1.3.16) and catalyze the following reaction : 4-hydroxy-2-oxoglutarate <=> pyruvate + glyoxylate. Furthermore, aldolases are provided that have HMG-aldolase activity (such as 4-hydroxy-4-methyl-2-oxoglutarate aldolase, pyruvate aldolase, aldolase gamma-methyl-gamma-hydroxy-alpha-ketoglutaric, 4-hydroxy-4- methyl-2-ketoglutarate aldolase, EC 4.1.3.17) and catalyze the following reaction: 4-hydroxy-4-methyl-2-oxoglutarate <=> 2-pyruvate. An HMG aldolase will also act on 4-hydroxy-4-methyl-2-oxoadipate and 4-carboxy-4-hydroxy-2-oxohexadioate.

     Aldolasa

R = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl

R2 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl

R3 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl, carboxylic acid.

Aldolases, such as a pyruvate aldolase, such as, but not limited to, an HMG and / or a KHG aldolase, are provided which facilitate the production of a 3,4-substituted 2-keto-glutarate. There is also disclosed a method of preparing a 3,4-substituted 2-keto glutarate comprising: (a) providing a polypeptide having an aldolase activity, such as a pyruvate aldolase activity, such as, non-exhaustively, an HMG aldolase and / or KMG aldolase activity; (b) providing a donor and an acceptor compound; and (c) contacting the polypeptide of step (a) with the compounds of step (b) under conditions in which aldolase catalyzes the synthesis of a 3,4-substituted 2-keto-glutarate, where optionally the donor and acceptor are a pyruvate or a pyruvate donor and an a-keto acid acceptor, a ketone and / or an aldehyde.

Aldolases are provided which facilitate the production of R-2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid (R-MP), a monatin precursor. A pyruvate aldolase, such as an HMG and / or a KHG aldolase, can be used in conjunction with a D-aminotransferase to prepare a 4-substituted D-glutamic acid or a derivative thereof. A 4-substituted Dglutamic acid and / or a derivative thereof can be used as an antibiotic, since these compounds have been found to inhibit bacterial glutamate racemase (WO0214261A3). Furthermore, a method for preparing a 4-substituted D-glutamic acid is disclosed comprising: (a) providing a polypeptide having an aldolase activity, such as a pyruvate aldolase activity, such as, non-exhaustively, a HMG aldolase and / or KMG aldolase activity; (b) providing an a-keto acid acceptor and a pyruvate or a pyruvate donor; and

(c) contacting the polypeptide of step (a) with the compounds of step (b) under conditions in which aldolase catalyzes the synthesis of a 4-substituted D-glutamic acid, where optionally the polypeptide has activity pyruvate aldolase, HMG aldolase and / or KHG aldolase and wherein optionally the method further comprises the use of a D-aminotransferase.

Isolated, synthetic or recombinant nucleic acids are disclosed comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% , 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or 100% (complete) sequence identity with respect to a nucleic acid disclosed herein, including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID N O: 69, SEQ ID

NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SE Q ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 33 7 and SEQ ID NO: 338 in a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500 or more waste. One or more nucleic acids encode at least one polypeptide that has aldolase activity, including pyruvate activity such as, non-exhaustively, HMG and / or KHG aldolase activity. In some embodiments, sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection.

Alternatively, one or more nucleic acids encode at least one polypeptide capable of generating an antibody that can specifically bind to a polypeptide of the invention, or these nucleic acids can be used as probes to identify or isolate aldolase encoding nucleic acids or to inhibit the expression of nucleic acids that express aldolase.

Nucleic acids can also include isolated, synthetic or recombinant nucleic acids that encode enzymes disclosed herein, such as enzymes that include one or more polypeptides that have a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO : 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140 , SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO : 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190 , SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO : 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240 , SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO : 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290 , SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO : 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 and SEQ ID NO: 334 and subsequences thereof, variants thereof and enzymatically active fragments thereof. The polypeptide may have aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity.

Nucleic acids encoding aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase preferably derived from mixed cultures are also disclosed. Furthermore, nucleic acids encoding an enzyme that forms or cleaves carbon-carbon bonds isolated from mixed cultures comprising polynucleotides as disclosed herein are described, such as a sequence having at least 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or more, or 100% (complete) sequence identity with respect to a nucleic acid disclosed herein such as SEQ ID NO: 1, SEQ ID NO : 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 , SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO : 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219 , SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 241, SEQ ID NO: 251, SEQ ID NO : 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269 , SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 291, SEQ ID NO: 301, SEQ ID NO : 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319 , SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 in a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more.

There are nucleic acids that encode aldolase enzyme, such as pyruvate aldolase enzyme, HMG and / or KHG enzyme, including polynucleotide sequences as disclosed herein and polypeptides encoding them, including enzymes disclosed herein, such as disclosed polypeptides herein, such as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96 , SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO : 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146 , SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO : 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196 , SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO : 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246 , SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO : 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296 , SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO : 330, SEQ ID NO: 332, or SEQ ID NO: 334, and enzymatically active fragments thereof, preferably derived from a common source, such

as an environmental source. Nucleic acids encoding aldolase enzyme, such as pyruvate aldolase enzyme, HMG and / or KHG enzyme preferably derived from environmental sources, such as mixed environmental sources, are also described.

In some embodiments, the sequence comparison algorithm is a BLAST version 2.2.2 algorithm, in which a filter setting of blastall -p blastp -d "nr pataa" -FF is set and all other options are configured with the default values.

Isolated, synthetic or recombinant nucleic acids are also disclosed including at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500 or more consecutive bases of a nucleic acid sequence disclosed herein, sequences basically identical to them and the sequences complementary thereto.

The isolated, synthetic, or recombinant nucleic acids disclosed herein encode a polypeptide that has aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, which is thermostable. The thermostable polypeptide disclosed herein can retain aldolase activity, such as pyruvate aldolase activity, such as HMG and / or KHG aldolase activity, under conditions comprising a temperature range of from about -100 ° C to about -80 ° C , approximately −80ºC to approximately −40ºC, approximately −40ºC to approximately −20ºC, approximately −20ºC to approximately 0ºC, approximately 0ºC to approximately 37ºC, approximately 0ºC to approximately 5ºC, approximately 5ºC to approximately 15ºC, approximately 15ºC to approximately 25ºC, approximately 25ºC at approximately 37ºC, approximately 37ºC to approximately 45ºC, approximately 45ºC to approximately 55ºC, approximately 55ºC to approximately 70ºC, approximately 70ºC to approximately 75ºC, approximately 75ºC to approximately 85ºC, approximately 85ºC to approximately 90ºC, approximately 90ºC to approximately 95ºC, approximately 95ºC to approximately Mind 100ºC, approximately 100ºC to approximately 105ºC, approximately 105ºC to approximately 110ºC, approximately 110ºC to approximately 120ºC or 95ºC, 96ºC, 97ºC, 98ºC, 99ºC, 100ºC, 101ºC, 102ºC, 103ºC, 104ºC, 105ºC, 106ºC, 107ºC, 108ºC, 109ºC , 110ºC, 111ºC, 112ºC, 113ºC, 114ºC, 115ºC or more. The thermostable polypeptides disclosed herein can retain an aldolase activity, such as a pyruvate aldolase activity, such as an HMG and / or KHG aldolase activity, at temperatures in the range of about -100 ° C to about -80 ° C, about - 80ºC to approximately −40ºC, approximately −40ºC to approximately −20ºC, approximately −20ºC to approximately 0ºC, approximately 0ºC to approximately 5ºC, approximately 5ºC to approximately 15ºC, approximately 15ºC to approximately 25ºC, approximately 25ºC to approximately 37ºC, approximately 37ºC to approximately 45ºC , approximately 45ºC to approximately 55ºC, approximately 55ºC to approximately 70ºC, approximately 70ºC to approximately 75ºC, approximately 75ºC to approximately 85ºC, approximately 85ºC to approximately 90ºC, approximately 90ºC to approximately 95ºC, approximately 95ºC to approximately 100ºC, approximately 100ºC to approximately 105ºC, approximately Accurately 105ºC to approximately 110ºC, approximately 110ºC to approximately 120ºC or 95ºC, 96ºC, 97ºC, 98ºC, 99ºC, 100ºC, 101ºC, 102ºC, 103ºC, 104ºC, 105ºC, 106ºC, 107ºC, 108ºC, 109ºC, 110ºC, 111ºC, 112ºC, 113ºC, 114ºC, 115ºC or more. The thermostable polypeptides disclosed herein retain aldolase activity at a temperature in the ranges described above, at about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, 0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

Isolated, synthetic, or recombinant nucleic acids encode a polypeptide that has aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, which is heat-tolerant. The thermotolerant polypeptides disclosed herein can retain an aldolase activity, such as a pyruvate aldolase activity, such as an HMG and / or KHG aldolase activity, after exposure to conditions comprising a temperature in the range of about - 100ºC to approximately −80ºC, approximately −80ºC to approximately −40ºC, approximately −40ºC to approximately −20ºC, approximately −20ºC to approximately 0ºC, approximately 0ºC to approximately 5ºC, approximately 5ºC to approximately 15ºC, approximately 15ºC to approximately 25ºC, approximately 25ºC to approximately 37ºC, approximately 37ºC to approximately 45ºC, approximately 45ºC to approximately 55ºC, approximately 55ºC to approximately 70ºC, approximately 70ºC to approximately 75ºC, approximately 75ºC to approximately 85ºC, approximately 85ºC to approximately 90ºC, approximately 90ºC to approximately 95ºC, approximately 95ºC to approximately 100ºC , approximately 100ºC to approximately 105ºC, approximately 105ºC to approximately 110ºC, approximately 110ºC to

approximately 120ºC or 95ºC, 96ºC, 97ºC, 98ºC, 99ºC, 100ºC, 101ºC, 102ºC, 103ºC, 104ºC, 105ºC, 106ºC, 107ºC, 108ºC, 109ºC, 110ºC, 111ºC, 112ºC, 113ºC, 114ºC, 115ºC or more. The thermotolerant polypeptides disclosed herein can retain an aldolase activity, such as a pyruvate aldolase activity, such as an HMG and / or KHG aldolase activity, after exposure to a temperature in the range of about −100 ° C to about −80ºC, approximately −80ºC to approximately −40ºC, approximately −40ºC to approximately −20ºC, approximately −20ºC to approximately 0ºC, approximately 0ºC to approximately 5ºC, approximately 5ºC to approximately 15ºC, approximately 15ºC to approximately 25ºC, approximately 25ºC to approximately 37ºC, approximately 37ºC to approximately 45ºC, approximately 45ºC to approximately 55ºC, approximately 55ºC to approximately 70ºC, approximately 70ºC to approximately 75ºC, approximately 75ºC to approximately 85ºC, approximately 85ºC to approximately 90ºC, approximately 90ºC to approximately 95ºC, approximately 95ºC to approximately 100ºC, approximately 100ºC aa approximately 105ºC, approximately 105ºC to approximately 110ºC, approximately 110ºC to approximately 120ºC or 95ºC, 96ºC, 97ºC, 98ºC, 99ºC, 100ºC, 101ºC, 102ºC, 103ºC, 104ºC, 105ºC, 106ºC, 107ºC, 108ºC, 109ºC, 110ºC, 111ºC, 112ºC , 113ºC, 114ºC, 115ºC or more. The thermotolerant polypeptides disclosed herein retain aldolase activity upon exposure to a temperature in the ranges described above, at about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, approximately pH 5.0, approximately pH 5.5, approximately pH 6.0, approximately pH 6.5, approximately pH 7.0, approximately pH 7.5, approximately pH 8.0, approximately pH 8.5, approximately pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

There are isolated, synthetic, or recombinant nucleic acids that comprise a sequence that hybridizes under stringent conditions to nucleic acids as disclosed herein including a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO : 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO : 105, SEQ ID NO: 107, SEQ ID NO: 1 09, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, or SEQ ID NO: 338, or fragments or subsequences thereof. Nucleic acids encode polypeptides that have aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity. Nucleic acids can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 , 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more residues in length or the full length of the gene or transcript. Strict conditions comprise a wash step comprising washing in 0.2X SSC at a temperature of about 65 ° C for about 15 minutes.

Nucleic acid probes exist to identify or isolate nucleic acids encoding polypeptides that have aldolase activity, including pyruvate activity such as, non-exhaustively, HMG and / or KHG aldolase activity, where the probes comprise about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more consecutive bases of a sequence as disclosed herein, and wherein the probes identify nucleic acid by binding or hybridization. The probes can comprise an oligonucleotide comprising between

about 10-100 consecutive bases of a sequence according to the invention or fragments or subsequences thereof, eg 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95 or 100 bases or more, or any desired intermediate length.

Nucleic acid probes exist to identify or isolate nucleic acids encoding polypeptides that have aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, where the probes comprise nucleic acids that they comprise a sequence of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 , 700, 750, 800, 850, 900, 950, 1000 or more residues of a nucleic acid as disclosed herein, such as a polynucleotide having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% (complete) of sequence identity with with respect to a nucleic acid disclosed herein. In some embodiments, sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection. The probes can comprise an oligonucleotide comprising between at least about 10-100 consecutive bases of a nucleic acid sequence disclosed herein or a subsequence thereof, eg 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 bases or more, or any desired intermediate length.

Pairs of amplification primers exist to amplify (such as by PCR) nucleic acids encoding polypeptides that have aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, where each pair primer is capable of amplifying a nucleic acid comprising a sequence as disclosed herein or fragments or subsequences thereof (see Sequence Listing). One or each member of the pair of amplification primer sequences may comprise an oligonucleotide comprising at least about 10 to 50, or more, consecutive bases of the sequence or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more consecutive bases in the sequence. Amplification primer pairs are also described, where each primer pair comprises a first member that has a sequence as established in approximately the first (5 ') 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more residues of a nucleic acid according to the invention, and a second member that has a sequence as established in approximately the first (5 ') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36 or more residues of the complementary strand of the first member.

There are nucleic acids encoding aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase generated by amplification, such as polymerase chain reaction (PCR), using a pair of amplification primers disclosed herein. Nucleic acids encoding aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are generated by amplification, such as polymerase chain reaction (PCR), using a pair of amplification primers as disclosed in the present. There are methods of preparing an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase by amplification, such as polymerase chain reaction (PCR), using a pair of amplification primers disclosed herein. The amplification primer pair amplifies a nucleic acid from a library, such as a genomic library, such as an environmental library.

There are methods of amplifying a nucleic acid encoding a polypeptide having aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, comprising amplifying a template nucleic acid with a pair of amplification primer sequences capable of amplifying a nucleic acid sequence disclosed herein or fragments or subsequences thereof.

There are expression cassettes that comprise a nucleic acid disclosed herein or a subsequence thereof. The expression cassette may comprise nucleic acid that is operably linked to a promoter. The promoter can be a viral, bacterial, mammalian, fungal, yeast or plant promoter. The plant promoter can be a potato, rice, corn, wheat, tobacco or barley promoter. The promoter can be a constitutive promoter. The constitutive promoter can comprise CaMV35S. The promoter can be an inducible promoter. The promoter can be a tissue specific promoter or a promoter regulated by the environment or by development. In this way, the promoter can be, for example, a seed-specific, leaf-specific, root-specific or stem-specific promoter or an abscission-induced promoter. The expression cassette may further comprise a plant or plant virus expression vector.

There are cloning vehicles that comprise an expression cassette (such as a vector) as disclosed herein or a nucleic acid as disclosed herein. The cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fossil, a bacteriophage, or an artificial chromosome. The viral vector may comprise an adenovirus vector, a retroviral vector, or an adeno-associated viral vector. The cloning vehicle may comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

Transformed cells comprising nucleic acids as disclosed herein or expression cassettes (such as vectors) disclosed herein or cloning vehicles disclosed herein are disclosed. The transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell, or a plant cell. The plant cell can be soybean, rapeseed, oilseed, tomato, sugar cane, cereal, potato, wheat, rice, corn, tobacco or barley cell.

There are transgenic non-human animals that comprise a nucleic acid as disclosed herein or an expression cassette (such as a vector) as disclosed herein. The animal is a mouse, a rat, a pig, a goat, or a sheep.

Transgenic plants comprising a nucleic acid as disclosed herein or an expression cassette (such as a vector) as disclosed herein are disclosed. The transgenic plant can be a cereal plant, a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant , a barley plant or a tobacco plant.

There are transgenic seeds that comprise a nucleic acid as disclosed herein or an expression cassette (such as a vector) as disclosed herein. The transgenic seed can be a cereal plant, a corn seed, a wheat grain, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a vegetable seed peanuts or tobacco.

There are antisense oligonucleotides that comprise nucleic acid sequences complementary to or capable of hybridizing under stringent conditions with nucleic acids as disclosed herein. Also disclosed are methods of inhibiting the translation of an enzyme aldolase message, such as pyruvate aldolase, HMG and / or KHG aldolase in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to, or capable of hybridizing under stringent conditions to a nucleic acid disclosed herein. The antisense oligonucleotide is from about 10 to about 50, about 20 to about 60, about 30 to about 70, about 40 to about 80, or about 60 to about 100 bases in length, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases in length.

Methods are disclosed for inhibiting translation of an enzyme aldolase message, such as pyruvate aldolase, such as enzyme HMG and / or KHG aldolase in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a sequence of Nucleic acids complementary to, or capable of hybridizing under stringent conditions to, a nucleic acid disclosed herein.

Double-stranded inhibitory RNA (RNAi or RNA) molecules (including small interfering RNA, or siRNA, to inhibit transcription and microRNA or miRNA, to inhibit translation) are also disclosed that comprise a subsequence of a sequence disclosed in the Present. In some cases, the siRNA is from about 21 to about 24 residues or from about at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more duplex nucleotides long. There are methods of inhibiting the expression of an aldolase enzyme, such as the pyruvate aldolase enzyme, such as HMG and / or KHG aldolase in a cell, comprising administering to the cell or expressing in the cell a double-stranded inhibitory RNA (siRNA or miRNA ), wherein the RNA comprises a subsequence of a sequence disclosed herein.

Also described are isolated, synthetic, or recombinant polypeptides that comprise an amino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76% , 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% (complete) of sequence identity with respect to a polypeptide or peptide disclosed herein in a region of about 10, 15 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300 , 325, 350 or more residues, or over the entire length of the polypeptide. In some embodiments, sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection. The polypeptide or peptide sequences disclosed herein include SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 , SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO : 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 , SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO : 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130 , S EQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID

NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, I KNOW Q ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 and SEQ ID NO: 334 and subsequences thereof, variants thereof and enzymatically active fragments thereof. The polypeptides disclosed herein also include fragments of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300 , 350, 400, 450, 500, 550, 600 or more residues in length or in the total length of an enzyme. The polypeptide or peptide sequences disclosed herein include sequence encoded by a nucleic acid disclosed herein. The polypeptide or peptide sequences disclosed herein include polypeptides or peptides specifically linked by an antibody disclosed herein (such as epitopes) or polypeptides or peptides that can generate an antibody disclosed herein (such as an immunogen).

In some embodiments, a polypeptide disclosed herein has at least one aldolase enzyme activity, such as, pyruvate aldolase enzyme activity, such as HMG and / or KHG aldolase. In other embodiments, a polynucleotide disclosed herein encodes a polypeptide having at least one aldolase enzyme activity, such as, pyruvate aldolase enzyme activity, such as HMG and / or KHG aldolase.

Also provided are isolated, synthetic, or recombinant polypeptides or peptides comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 , 100, 125, 150 or more consecutive bases of the polypeptide or peptide sequences disclosed herein, sequences basically identical thereto and sequences complementary thereto. The peptide can be, for example, an immunogenic fragment, a motif (such as a binding site), a signal sequence, a prepro sequence, or an active site.

The disclosure describes isolated, synthetic or recombinant nucleic acids comprising a sequence encoding a polypeptide having an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, and a signal sequence, wherein the nucleic acid it comprises a sequence disclosed herein. A "signal sequence" means a secretory signal or other domain that facilitates the secretion of the aldolase disclosed herein in the host cell. The signal sequence can be derived from another aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, or a non-aldolase enzyme, such as non-pyruvate aldolase, such as non-HMG and / or non-KHG aldolase (heterologous). The disclosure further discloses isolated, synthetic, or recombinant nucleic acids comprising a sequence encoding a polypeptide having an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, wherein the sequence does not contain a signal sequence. and the nucleic acid comprises a sequence disclosed herein. The disclosure discloses isolated, synthetic, or recombinant polypeptides comprising polypeptides disclosed herein that lack all or part of a signal sequence. In some embodiments, the isolated, synthetic, or recombinant polypeptide may comprise the polypeptide disclosed herein comprising a heterologous signal sequence, such as an aldolase enzyme signal sequence, such as pyruvate aldolase, such as HMG and / or KHG aldolase, or non-aldolase enzyme signal sequence, such as non-pyruvate aldolase, such as non-HMG and / or non-KHG aldolase, heterologous.

The disclosure further discloses chimeric proteins comprising a first domain comprising a signal sequence disclosed herein and at least a second domain. The protein can be a fusion protein. The second domain can comprise an enzyme. Protein can be a non-enzyme.

The disclosure discloses chimeric polypeptides comprising at least one first domain comprising signal peptide (SP), a prepro sequence and / or a catalytic domain (CD) disclosed herein and at least one second domain comprising a heterologous peptide or polypeptide, wherein the heterologous peptide or polypeptide is not naturally associated with the signal peptide (SP), prepro sequence, and / or catalytic domain (CD). In some embodiments, the heterologous peptide or polypeptide is not an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase. The heterologous peptide or polypeptide can be at the amino terminal, carboxy terminal, or both ends of the signal peptide (SP), the prepro sequence, and / or the catalytic domain (CD).

The disclosure discloses isolated, synthetic or recombinant nucleic acids encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide (SP), a prepro domain and / or a catalytic domain (CD) disclosed in the present and at least a second domain that

it comprises a heterologous peptide or polypeptide, wherein the heterologous peptide or polypeptide does not naturally associate with the signal peptide (SP), prepro domain and / or catalytic domain (CD).

The disclosure discloses isolated, synthetic, or recombinant signal sequences (such as signal peptides) consisting of or comprising a sequence as set forth in residues 1-14, 1-15, 1-16, 1-17, 1-18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1-45, 1-46 or 1-47, of a polypeptide disclosed herein such as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 7 2, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ I D NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO : 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240 , SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO : 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290 , SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO : 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334. In some embodiments, the disclosure discloses signal sequences comprising the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino terminal residues of a polypeptide disclosed herein.

Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase can comprise a specific activity of from about 10 to about 12,000 units per milligram of protein. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can also comprise a specific activity of from about 1,000 to about 10,000 units per milligram of protein, or from about 5,000 to about 7,500 units per milligram of protein . Alternatively, the activity of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, comprises a specific activity in the range of from about 10 to about 7,500 units per milligram of protein, or from about 5,000 to about 12,000 units per milligram of protein. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can comprise a specific activity in the range of from about 10 to about 5000 units per milligram of protein, or from about 7500 to about 10,000 units per milligram of protein. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can comprise a specific activity in the range of from about 10 to about 2500 units per milligram of protein. Alternatively, the activity of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, comprises a specific activity in the range of from about 10 to about 1000 units per milligram of protein. An exemplary method of measuring the activity of different aldolase enzymes, such as pyruvate aldolases, such as HMG and / or KHG aldolase, uses a general substrate, 4-carboxy-4-hydroxy-2-oxoadipate ("CHA"). A typical assay comprises 50mM sodium phosphate pH 7.5, 1mM MgCl2, 1mM CHA, 10 µg / ml D-lactate dehydrogenase (“LDH”) from Lactobacillus leichmanii (Sigma-Aldrich, St. Louis, MO) 0.5 mM NADH. The assay is started by adding the enzyme to be measured. Pyruvate release, along with NAD + formation, is continuously monitored on a spectrophotometer at 340 nm. A unit of enzyme activity is defined as the amount that releases enough pyruvate to decrease the absorbance by 1 OD per minute at 340 nm.

Thermo tolerance of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, may comprise the retention of at least half of the specific activity of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase after heating to an elevated temperature, such as a temperature of about 0 ° C to about 20 ° C, about 20 ° C to about 37 ° C, about 37 ° C to about 50 ° C, about 50 ° C to about 70 ° C,

approximately 70ºC to approximately 75ºC, approximately 75ºC to approximately 80ºC, approximately 80ºC to approximately 85ºC, approximately 85ºC to approximately 90ºC, approximately 90ºC to approximately 95ºC, approximately 95ºC to approximately 100ºC, approximately 100ºC to approximately 110ºC or higher. Thermotolerance may also comprise the retention of specific activity of from about 10 to about 12,000 units per milligram of protein, or from about 5000 to about 10,000 units per milligram of protein, after heating to an elevated temperature, as described above. Thermotolerance may also comprise the retention of specific activity in the range of from about 10 to about 5000 units per milligram of protein after heating to an elevated temperature, as described above.

The disclosure discloses isolated, synthetic, or recombinant polypeptides disclosed herein, wherein the polypeptides comprise at least one glycosylation site. In some embodiments, the glycosylation can be an N-linked glycosylation. The polypeptide can be glycosylated after being expressed in a P. pastoris or S. pombe host or in a mammalian host cell.

The polypeptide can retain aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, under conditions having a pH of about 6.5, pH 6, pH 5.5, pH 5, pH 4.5 , pH 4.0, pH 3.5, pH 3.0 or less (more acidic). The polypeptide can also retain an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, under conditions having a pH of about 7, pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 or more (more basic). The polypeptide can retain an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, after exposure to conditions having a pH of about 6.5, pH 6, pH 5.5, pH 5 , pH 4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic). The polypeptide may also retain an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, after exposure to conditions having a pH of about 7, pH 7.5, pH 8.0, pH 8 , 5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 or more (more basic).

The aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein has activity under alkaline conditions, such as alkaline conditions of the intestine, such as the small intestine. The polypeptide can retain activity after exposure to the acidic pH of the stomach.

The disclosure discloses protein preparations comprising a polypeptide (including peptides) disclosed herein, wherein the protein preparation comprises a liquid, a solid, or a gel. The disclosure discloses heterodimers comprising a polypeptide disclosed herein and a second member, such as a polypeptide or another (second) domain. The second member of the heterodimer may be a different aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, a different enzyme, or another protein. The second domain can be a polypeptide and the heterodimer can be a fusion protein. The second domain can also be an epitope or a tag. The disclosure further discloses homomultimers, including, but not limited to, homodimers, homotrimers, homotetramers, homopentamers, and homohexamers, which comprise a polypeptide disclosed herein.

The disclosure discloses immobilized polypeptides (including peptides) having aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, wherein the immobilized polypeptide comprises a polypeptide disclosed herein, a nucleic acid encoded polypeptide disclosed herein or a polypeptide comprising a polypeptide disclosed herein and a second domain. The polypeptide can be immobilized in a cell, metal, resin, polymer, ceramic, glass, microelectrode, graphite particle, bead, gel, plate, array, or capillary tube.

The disclosure further discloses assays comprising an immobilized nucleic acid disclosed herein, such as probes disclosed herein. The disclosure also discloses assays comprising an antibody disclosed herein.

The disclosure discloses isolated, synthetic or recombinant antibodies that specifically bind to a polypeptide disclosed herein or to a nucleic acid encoded polypeptide disclosed herein. These antibodies disclosed herein can be a monoclonal or polyclonal antibody. Hybridomas comprising an antibody disclosed herein are also described, such as an antibody that specifically binds to a polypeptide disclosed herein or a nucleic acid encoded polypeptide disclosed herein. The disclosure also discloses nucleic acids encoding these antibodies.

The disclosure discloses methods for isolating or identifying polypeptides having aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, comprising the steps of: (a) providing an antibody disclosed herein; (b) providing a sample comprising polypeptides; and (c) contacting the sample from step (b) with the antibody from step (a) under conditions in which the antibody can specifically bind to the polypeptide and thereby isolate or identify a polypeptide having an activity of aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase.

Methods of preparing an anti-aldolase enzyme antibody, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase are disclosed comprising administering to a non-human animal a nucleic acid disclosed herein or a disclosed polypeptide herein or subsequences thereof in an amount sufficient to generate a humoral immune response and thereby make an anti-aldolase enzyme antibody, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase. Also disclosed are methods of preparing an anti-aldolase immune response, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase (cellular or humoral) which comprises administering to a non-human animal a nucleic acid disclosed in the present or a polypeptide disclosed herein or subsequences thereof in an amount sufficient to generate an immune response (cellular or humoral).

Methods of producing a recombinant polypeptide are described comprising the steps of: (a) providing a nucleic acid disclosed herein operably linked to a promoter; and (b) expressing the nucleic acid from step (a) under conditions that allow expression of the polypeptide and thereby produce a recombinant polypeptide. In some embodiments, the method may further comprise transforming a host cell with the nucleic acid from step (a) and then expressing the nucleic acid from step (a) and thereby producing a recombinant polypeptide in a transformed cell.

Methods for identifying a polypeptide having aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are described, comprising the following steps: (a) providing a polypeptide disclosed herein; or a nucleic acid encoded polypeptide disclosed herein; (b) providing aldolase enzyme substrate, such as pyruvate aldolase, such as HMG and / or KHG aldolase; and (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a product. reaction, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The substrate can be a carbohydrate, a compound comprising a carbohydrate and / or a carbohydrate mimetic.

Methods for identifying aldolase enzyme substrate, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are described comprising the following steps: (a) providing a polypeptide disclosed herein or nucleic acid encoded herein. Present; (b) provide a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as an aldolase enzyme substrate, such as pyruvate aldolase, HMG and / or KHG aldolase.

Methods are described for determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under permissive conditions to transfer the nucleic acid to a polypeptide, in where the nucleic acid comprises a nucleic acid disclosed herein, or providing a polypeptide disclosed herein; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.

Methods for identifying a modulator of an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are described, comprising the following steps: (a) providing a polypeptide disclosed herein or a polypeptide encoded by a nucleic acid disclosed herein; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, wherein A change in enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, measured in the presence of the test compound compared to activity in the absence of the test compound determines that the test compound modulates the activity of aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be measured by providing an aldolase enzyme substrate, such as pyruvate aldolase, HMG and / or KHG aldolase and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or an increase in the amount of the substrate or a decrease in the amount of a reaction product. A decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of the activity of aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase. An increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of the activity of aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase.

Computer systems comprising a processor and a data storage device are described, wherein said data storage device has stored a polypeptide sequence or nucleic acid sequence disclosed herein (such as a polypeptide or peptide encoded by a nucleic acid disclosed herein). The computer system may further comprise a comparison algorithm.

of sequences and a data storage device having at least one reference sequence stored therein. The sequence comparison algorithm may comprise a computer program that indicates polymorphisms. The computer system may further comprise an identifier that identifies one or more characteristics in said sequence. Computer readable media are also disclosed having stored a polypeptide sequence or a nucleic acid sequence disclosed herein. Methods for identifying a characteristic in a sequence are disclosed comprising the steps of: (a) reading the sequence using a computer program that identifies one or more characteristics in a sequence, wherein the sequence comprises a polypeptide sequence or an acid sequence nucleic acids disclosed herein; and (b) identify one or more characteristics in the sequence with the computer program. Methods for comparing a first sequence with a second sequence are disclosed comprising the steps of: (a) reading the first sequence and the second sequence through the use of a sequence comparing computer program, wherein the first sequence comprises a sequence of polypeptides or a nucleic acid sequence disclosed herein; and (b) determine differences between the first sequence and the second sequence with the computer program. The step of determining differences between the first sequence and the second sequence may further comprise the step of identifying polymorphisms. The method may further comprise an identifier that identifies one or more characteristics in a sequence. The method may comprise reading the first sequence using a computer program and identifying one or more features in the sequence.

Methods are disclosed for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, from a sample, such as an environmental sample, comprising the steps of: (a) providing a pair of amplification primer sequences to amplify a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, wherein the pair of primers it is capable of amplifying a nucleic acid disclosed herein; (b) isolating a nucleic acid from the sample or treating the sample such that the nucleic acid in the sample is accessible for hybridization with the pair of amplification primers; and (c) combining the nucleic acid from step (b) with the pair of amplification primers from step

(to)

 and amplifying nucleic acid from the sample and thereby isolating or recovering a nucleic acid encoding a polypeptide having an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, from a sample. One or each member of the pair of amplification primer sequences may comprise an oligonucleotide comprising a pair of amplification primer sequences disclosed herein, eg with at least about 10 to 50 consecutive bases of a sequence disclosed herein. The sample may be an environmental sample.

Methods are disclosed for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, from a sample, such as an environmental sample, comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid disclosed herein or a subsequence thereof; (b) isolating a nucleic acid from the sample

or treating the sample such that the nucleic acid in the sample is accessible for hybridization with a polynucleotide probe from step (a); (c) combining the isolated nucleic acid or the treated test from step (b) with the polynucleotide probe from step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a) and thereby isolating or recovering a nucleic acid encoding a polypeptide having an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, from a sample. The sample may comprise a water sample, a liquid sample, a soil sample, an air sample, or a biological sample. The biological sample can be derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a mammalian cell. The sample may be an environmental sample.

Methods for generating a variant of a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are disclosed, comprising the steps of: (a) providing a nucleic acid template comprising a nucleic acid disclosed herein; and

(b)

 modify, delete or add one or more nucleotides in the template sequence or a combination thereof, to generate a variant of the template nucleic acid. The method may further comprise expressing the variant nucleic acid to generate a variant aldolase, such as pyruvate aldolase enzyme polypeptide, such as HMG and / or KHG aldolase. Modifications, additions, or deletions can be introduced by a method comprising error-prone PCR, transposition, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, ensemble mutagenesis exponential, site specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), Chromosome Saturation Mutagenesis (CSM) or a combination thereof. Alternatively, modifications, additions, or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, discontinuous duplex mutagenesis, point mismatch repair mutagenesis , poorly repairing host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, creation of chimeric nucleic acid multimers and a combination of the same.

The method may be repeated until an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase having altered or different activity or altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced. The aldolase enzyme polypeptide, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase, variant may be thermotolerant and retain some activity after exposure to an elevated temperature. The aldolase variant, such as pyruvate aldolase enzyme polypeptide, such as HMG and / or KHG aldolase may have increased glycosylation compared to the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase encoded by a nucleic acid template. Alternatively, the aldolase polypeptide, such as pyruvate aldolase, such as HMG and / or KHG aldolase, variant has an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, at a high temperature, wherein the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase encoded by the template nucleic acid is not active at high temperatures. The method can also be repeated until an aldolase enzyme coding sequence occurs, such as pyruvate aldolase, HMG and / or KHG aldolase having altered codon usage relative to that of template nucleic acid. The method can also be repeated until an aldolase enzyme gene, such as pyruvate aldolase, HMG and / or KHG aldolase, is produced that has a higher or lower level of message expression or stability than the template nucleic acid.

Methods are described for modifying codons in a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, to increase its expression in a host cell, said method comprising the following steps: (a) providing a nucleic acid disclosed herein that encodes a polypeptide having an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase; and (b) identifying a non-preferred or less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon overrepresented in gene encoding sequences in the host cell and a non-preferred or less preferred codon is an underrepresented codon in gene encoding sequences in the host cell and thereby modify nucleic acid to increase its expression in a host cell .

Methods are disclosed for modifying codons in a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, said method comprising the following steps: (a) providing a nucleic acid disclosed herein; and (b) identifying a codon in the nucleic acid from step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon and thereby modifying codons in a nucleic acid encoding an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase.

Methods are disclosed for modifying codons in a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, to increase its expression in a host cell, said method comprising the following Steps: (a) providing a nucleic acid disclosed herein that encodes an aldolase enzyme polypeptide, such as pyruvate aldolase, HMG and / or KHG aldolase; and (b) identifying a non-preferred or less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon overrepresented in gene encoding sequences in the host cell and a non-preferred or less preferred codon is an underrepresented codon in gene encoding sequences in the host cell and thereby modify nucleic acid to increase its expression in a host cell .

Methods are disclosed for modifying a codon in a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, to decrease its expression in a host cell, said method comprising the following steps: (a) providing a nucleic acid disclosed herein; and (b) identify at least one preferred codon in the nucleic acid of step (a) and replace it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is an over-represented codon in gene coding sequences in a host cell and a non-preferred or less preferred codon is an underrepresented codon in gene coding sequences in the host cell and thereby modify nucleic acid to decrease its expression in a host cell. The host cell may be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell, or a mammalian cell.

Methods for producing a nucleic acid library encoding a plurality of active sites or substrate binding sites of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are disclosed, wherein the active sites or sites Substrate binding techniques are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site, said method comprising the following steps: (a) providing a first nucleic acid encoding a first site active or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hydrides under stringent conditions with a nucleic acid disclosed herein, and the nucleic acid encodes an active site of an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase or an aldolase enzyme substrate binding site, such as pyruvate aldolase, HMG and / or KHG aldolase; (b) provide a

set of mutagenic oligonucleotides that encode natural amino acid variants in a plurality of target codons in the first nucleic acid; and (c) using the set of mutagenic oligonucleotides to generate a set of active site coding or substrate binding site coding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized and that way produce a library of nucleic acids encoding a plurality of enzyme aldolase active sites or substrate binding sites, such as pyruvate aldolase, such as HMG and / or modified KHG aldolase. The method may comprise mutagenizing the first nucleic acid of step (a) by a method comprising an optimized targeted evolution system, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), error-susceptible PCR , transposition, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive assembly mutagenesis, exponential assembly mutagenesis, site-specific mutagenesis, gene reassembly, and a combination thereof. The method may also comprise mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, batch duplex mutagenesis, mutagenesis point mismatch repair gene, poorly repaired host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction purification mutagenesis, artificial gene synthesis, pool mutagenesis, acid multimer creation chimeric nucleic acids and a combination thereof.

Methods for obtaining a small molecule are also disclosed, comprising the following steps: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase encoded by a nucleic acid disclosed herein; (b) providing a substrate for at least one of the enzymes in step (a); and (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions. Methods for modifying a small molecule are further described which comprise the following steps: (a) providing an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, wherein the enzyme comprises a polypeptide disclosed herein, or a polypeptide encoded by a nucleic acid disclosed herein or a subsequence thereof; (b) providing a small molecule; and (c) reacting the enzyme from step (a) with the small molecule from step (b) under conditions that facilitate an enzyme reaction catalyzed by the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase. and thereby modifying a small molecule by an enzymatic aldolase reaction, such as pyruvate aldolase, HMG and / or KHG aldolase. The method may comprise a plurality of small molecule substrates for the enzyme in step (a) and thereby generate a library of modified small molecules produced by at least one enzyme reaction catalyzed by the enzyme aldolase, such as pyruvate aldolase, such such as HMG and / or KHG aldolase. The method may comprise a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions. The method may further comprise the step of evaluating the library to determine if a particular modified small molecule that exhibits a desired activity is present in the library. The library evaluation step may further comprise the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by evaluating the portion of the modified small molecule to detecting the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.

Methods for determining a functional fragment of an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, are described comprising the steps of: (a) providing an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, wherein the enzyme comprises a polypeptide disclosed herein or a nucleic acid encoded polypeptide disclosed herein or a subsequence thereof; and (b) removing a plurality of amino acid residues from the sequence of step (a) and evaluating the remaining subsequence to detect an activity of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase and thus determining a functional fragment of an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase is measured by providing an aldolase enzyme substrate, such as pyruvate aldolase, HMG and / or KHG aldolase and detecting a decrease in the amount of the substrate or a increase in the amount of a reaction product.

Methods for manipulating whole cells of new or modified phenotypes using real-time metabolic flow analysis are disclosed, said method comprising the following steps: (a) obtaining a modified cell by modifying the genetic composition of a cell, where the genetic composition is modified by the addition to the cell of a nucleic acid according to the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and (d) analyze the data from step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, and thereby identify a manipulated phenotype in the cell using metabolic flow analysis. in real time. In some

In embodiments, the genetic makeup of the cell can be modified by a method that comprises deleting a sequence or modifying a sequence in the cell, or disabling expression of a gene. The method may further comprise selecting a cell that comprises a newly manipulated phenotype. The method may comprise culturing the selected cell and thus generating a new cell strain comprising a newly manipulated phenotype.

Methods are described for increasing the heat tolerance or thermostability of an aldolase enzyme polypeptide, such as pyruvate aldolase, HMG and / or KHG aldolase, the glycosylating method comprising an aldolase enzyme polypeptide, such as pyruvate aldolase, HMG and / or KHG aldolase, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide disclosed herein; or a polypeptide encoded by a nucleic acid sequence disclosed herein and thereby increase the heat tolerance or thermostability of the aldolase polypeptide, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The specific activity of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase can be thermostable or thermotolerant at a temperature in the range of from more than about 37 ° C to about 95 ° C.

Methods are disclosed for overexpressing an aldolase polypeptide, such as pyruvate aldolase, such as HMG and / or KHG aldolase, in a cell comprising expressing a vector comprising a nucleic acid comprising a nucleic acid disclosed herein or a sequence of nucleic acids disclosed herein, where sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection, where overexpression is carried out by using a high activity promoter, a vector dicistronic or by amplification of the vector gene.

Methods for preparing a transgenic plant are disclosed comprising the following steps: (a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a nucleic acid sequence disclosed herein and thereby produce a transformed plant cell; and

(b) produce a transgenic plant from the transformed cell. Step (a) may further comprise introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. Step (a) may further comprise introducing the heterologous nucleic acid sequence directly into the plant tissue by bombardment with DNA particles. Alternatively, step (a) may further comprise introducing the heterologous nucleic acid sequence into the DNA of the plant cell using an Agrobacterium tumefaciens host. The plant cell may be a sugarcane, beet, soy, tomato, potato, corn, rice, wheat, tobacco or barley cell.

Methods are also disclosed for expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence it comprises a nucleic acid disclosed herein; (b) cultivating the plant under conditions in which the heterologous nucleic acid sequence is expressed in the plant cell. Methods are described for expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence disclosed herein; (b) cultivating the plant under conditions in which the heterologous nucleic acid sequence is expressed in the plant cell.

The invention provides food or feed comprising a polypeptide disclosed herein or a nucleic acid encoded polypeptide disclosed herein. Also disclosed are foods, feed, liquids, such as beverages (such as fruit juices or beer), breads or doughs, or baked goods or beverage precursors (such as wort), which comprise a polypeptide disclosed herein. Further described are additives for food, feed or beverages comprising a polypeptide disclosed herein. Food or nutritional supplements, such as for a human or an animal, are also disclosed that comprise a polypeptide disclosed herein, such as a nucleic acid encoded polypeptide disclosed herein.

The polypeptide in the food or nutritional supplement can be glycosylated. Edible enzyme delivery matrices are also disclosed that comprise a polypeptide disclosed herein, such as a nucleic acid encoded polypeptide disclosed herein. The delivery matrix can comprise a granulate. The polypeptide can be glycosylated. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase may be heat tolerant. Aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase can be thermostable.

Food, feed or nutritional supplements are disclosed that comprise a polypeptide disclosed herein. Methods are described for using an enzyme aldolase, such as pyruvate aldolase, HMG and / or KHG aldolase as a nutritional supplement in an animal diet, said method comprising: preparing a nutritional supplement containing an enzyme aldolase, such as pyruvate aldolase, HMG and / or KHG aldolase comprising at least thirty contiguous amino acids of a polypeptide disclosed herein; and administer the nutritional supplement to an animal. The animal can be a human, a ruminant, or a monogastric animal. The aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase can be prepared by expression

of a polynucleotide encoding the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase in an organism selected from the group consisting of a bacterium, a yeast, a plant, an insect, a fungus, and an animal . The organism can be selected from the group consisting of S. pombe, S. cerevisiae, Pichia pastoris, E. coli, Streptomyces sp., Bacillus sp. Pseudomonas sp., Aspergillus sp. and Lactobacillus sp.

Edible enzyme delivery matrices are disclosed comprising a thermostable recombinant aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, such as a polypeptide disclosed herein. Methods for administering an enzyme aldolase supplement such as pyruvate aldolase, HMG and / or KHG aldolase to an animal are disclosed, said method comprising: preparing an edible enzyme delivery matrix in the form of granules comprising a granulated edible carrier and a Recombinant thermostable aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, where the granules readily disperse the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase contained therein in the aqueous medium , and administer the edible enzyme delivery matrix to the animal. The recombinant aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase can comprise a polypeptide disclosed herein. The aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be glucoseed to provide thermostability under the granulating conditions. The delivery matrix can be formed by granulating a mixture comprising a grain germ and an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase. Granulation conditions may include steam application. Granulation conditions may comprise applying a temperature above about 80 ° C for about 5 minutes and the enzyme retains a specific activity of at least 350 to about 900 units per milligram of enzyme.

Further disclosed are pharmaceutical compositions comprising an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase disclosed herein or a nucleic acid encoded polypeptide disclosed herein. The pharmaceutical composition can act as a digestive aid.

A compound containing a carbon-carbon bond is in contact with a polypeptide disclosed herein that has enzyme aldolase activity, such as, pyruvate aldolase enzyme activity, such as HMG and / or KHG aldolase, at a pH in the range from about pH 3.0 to about 9.0, about 3.0 to about 10.0, about 3.0 to about 11.0 or more. A compound containing a carbon-carbon bond is in contact with the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, at a temperature of at least about 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85ºC, 90ºC or more.

This disclosure provides, among other things, polypeptides that are useful in facilitating a process reaction to produce monatin, monatin derivatives, and salts thereof, for example in the production of R-2-hydroxy 2- (indole-3-ylmethyl acid). ) -4-ketoglutaric (also known as R-alpha keto acid monatin, precursor of Rmonatin, R-MP, and the alpha-keto form of monatin), a precursor to certain monatin stereoisomers, such as (R, R) - and (S, R) -monatin. The disclosure also provides methods for preparing monatin, monatin derivatives and salts, and internal condensation products thereof using one or more polypeptides disclosed herein. Methods for synthesizing R-MP, monatin stereoisomers and / or monatin derivative stereoisomers include the use of one or more aldolase-active polypeptides of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ I D NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO : 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148 , SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO : 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198 , SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO : 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248 , SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID

NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 and SEQ ID NO: 334, and enzymatically active fragments thereof.

Also, methods for synthesizing R-MP, monatin stereoisomers and / or monatin derivative stereoisomers can include the use of a polypeptide with aldolase activity encoded by a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or 100% ( complete) of sequence identity with respect to the nucleic acid disclosed herein including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 1 89, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 211, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 in a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500 or more waste.

Furthermore, methods for synthesizing R-MP, monatin stereoisomers and / or monatin derivative stereoisomers may include the use of an aldolase-active polypeptide encoded by a nucleic acid sequence that is hydrolyzed under stringent conditions with a nucleic acid SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO : 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 , SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO : 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99 , SEQ ID NO : 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117 , SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO : 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167 , SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO : 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217 , SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO : 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267 , SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299,

SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338.

The invention provides a method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, wherein a reaction in the pathway is facilitated by one or more polypeptides selected from isolated or recombinant polypeptides comprising the amino acid sequence of SEQ ID NO: 276 or fragments or subsequences thereof having aldolase activity as defined in the claims. In some embodiments, the fragments or subsequences thereof have an aldolase activity of at least 0.2 mg MP / mg protein / h. In other embodiments, the fragments or subsequences thereof have an aldolase activity of at least 0.1 mg MP / mg protein / h. In some embodiments, the reaction facilitated by one or more polypeptides disclosed herein is carried out in about 1.0 to about 10.0 mM MgCl2. In other embodiments, the reaction facilitated by one or more polypeptides disclosed herein is carried out at about pH 7.0 to about pH 11.5. Even in other embodiments, the reaction facilitated by one or more polypeptides disclosed herein is carried out in about 0.005% to about 1% polysorbate detergent.

In some embodiments, the reaction is a reaction between indole-3-pyruvate and a C3 carbon source. In some embodiments, the reaction preferably produces R-2-hydroxy-2- (indole-3-yl-methyl) -4-ketoglutaric acid instead of S-2-hydroxy-2- (indole-3-yl-methyl) acid. ) -4-ketoglutaric.

In some embodiments, the product obtained by the multistage route is (R, R) -monatin, salts thereof, and combinations thereof.

In other embodiments, (R, R) -monatin is produced in larger amounts than (S, S) -monatin, (S, R) -monatin

or (R, S) -monatin.

The invention provides a method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, wherein a Reaction in the pathway is facilitated by at least one polypeptide encoded by a nucleic acid sequence comprising a sequence having a sequence identity percentage of at least 95% with respect to SEQ ID NO: 275. In other embodiments, the percentage of sequence identity is 100%.

The invention provides a method comprising a reaction that preferably produces R-2-hydroxy-2 (indole-3-yl-methyl) -4-ketoglutaric acid instead of S-2-hydroxy-2- (indole-3- yl-methyl) -4-ketoglutaric wherein a polypeptide encoded by a nucleic acid sequence comprising a sequence having at least 95% sequence identity to SEQ ID NO: 275 is used to facilitate a one-way reaction multi-stage.

The invention provides a method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, wherein a Reaction in the pathway is facilitated by at least one polypeptide encoded by a nucleic acid sequence comprising a sequence that remains hybridized under stringent conditions to a nucleic acid of SEQ ID NO: 275 as defined in the claims.

The invention also provides a method comprising: producing a chosen product of R-2-hydroxy 2 (indole-3-ylmethyl) -4-ketoglutaric acid, salts thereof and combinations thereof in one way, wherein a reaction in the pathway is facilitated by one or more polypeptides chosen from isolated or recombinant polypeptides comprising the amino acid sequence of SEQ ID NO: 276 or fragments or subsequences thereof having aldolase activity, wherein said R-2-hydroxy acid 2- (indole-3-ylmethyl) -4-ketoglutaric, salts thereof and combinations thereof is used to prepare a sweetening product.

A method is further described which comprises: producing a chosen monatin precursor product, salts thereof and combinations thereof in a multi-step pathway, wherein a pathway reaction is facilitated by at least one polypeptide encoded by a sequence of nucleic acids comprising a sequence having a sequence identity percentage of at least 50% with respect to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 , SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121,

SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, or SEQ ID NO: 338, where said monatin precursor, salts thereof and combinations thereof is sweet.

Also disclosed is a method comprising: producing a chosen monatin precursor product, salts thereof, and combinations thereof in a multi-step pathway, wherein a pathway reaction is facilitated by at least one polypeptide encoded by a sequence of nucleic acids comprising a sequence that hydrides under stringent conditions with a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO : 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO : 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79 , SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 301, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 or SEQ ID NO: 338, wherein said monatin precursor, salts thereof, and combinations thereof It is sweet.

In an effort to be concise, whenever it is identified that intermediates / products have been formed in the specification and claims (such as monatin, monatin precursor or monatin derivative or derivatives), it should be understood that the expression "and / or or leave the same "where appropriate. In other words, for example, the phrase "indole-3-pyruvate converts to MP" should be understood to include "indole-3-pyruvic acid converts to MP and / or salts thereof". The person skilled in the art will, in fact, appreciate that under the described reaction conditions, the salts of the intermediates / products are in fact present.

According to some embodiments, the method produces a monatin or monatin derived composition wherein the monatin or monatin derived component of the composition includes only the R, R and S, R forms of monatin or monatin derivative. The term "alone", when used to indicate that only certain isomers are formed, means that the pathway would produce only the identified isomers if racemization does not occur. Accordingly, the term "alone" does not mean the absence of other isomers, but the person skilled in the art will understand that other isomeric forms may be present in a relatively small amount due to the racemization that may occur. According to some embodiments, the method produces a composition wherein the monatin or monatin-derived component of the composition includes only the R, R form of monatin or

monatin derivative (unless the point is reached that racemization occurs resulting in other isomeric forms).

As used herein, the phrase "monatin composition" means compositions that include one or more monatin isomers; the expression may also mean only a simple isomeric form of monatin and nothing else.

As used herein, the phrase "monatin-derived composition" means compositions that include one or more isomers of a monatin derivative; the expression may also mean only a simple isomeric form of the monatin derivative and nothing else.

As used herein, the phrase "monatin derivative" has the following structure:

wherein Ra, Rb, Rc, Rd and Re each independently represent any substituent that is selected from a hydrogen atom, a hydroxyl group, a C1-C3 alkyl group, a C1-C3 alkoxy group, an amino group or an atom halogen, such as an iodine atom, bromine atom, chlorine atom, or fluorine atom. However, Ra, Rb, Rc, Rd, and Re cannot all simultaneously be hydrogen. Alternatively, Rb andRc, and / or Rd andRe together can form a C1-C4 alkylene group, respectively.

As used herein, "substituted indole-3-pyruvate" means that one or more carbon atoms in the indole ring of indole-3-pyruvate is independently substituted by one or more of the substituent groups Ra, Rb, Rc, Rd, and Re defined above. However, Ra, Rb, Rc, Rd, and Re cannot all simultaneously be hydrogen. Alternatively, Rb and Rc, and / or Rd and Re together can form a C1-C4 alkylene group, respectively.

As used herein, "substituted tryptophan" means that one or more carbon atoms of the indole ring of tryptophan is independently substituted by one or more of the substituent groups Ra, Rb, Rc, Rd, and Re defined above. However, Ra, Rb, Rc, Rd, and Re cannot all simultaneously be hydrogen. Alternatively, Rb andRc, and / or Rd andRe together can form a C1-C4 alkylene group, respectively. In one embodiment, the substituted tryptophan contains the or the same substituent groups on the indole ring as the final monatin derivative.

Furthermore, the biosynthetic pathways for producing monatin described herein can use a substituted tryptophan to provide monatin derivatives that are likely to be sweet. In some embodiments, the substituted tryptophan to be used in the biosynthetic pathways described herein include tryptophan and chlorinated 5-hydroxytryptophan.

For example, chlorinated D-tryptophan, which has structural similarities to (R, R) -monatin, have been identified as non-nutritive sweeteners (particularly 6-chloro-D-tryptophan). Similarly, hydroxy-substituted and halogenated monatin forms have been found to be sweet. See United States Patent Application Published No. 2005/0118317. Halogen and hydroxyl groups can be replaced by hydrogen, particularly at positions 1-4 of the benzene ring in the indole of tryptophan, without interfering in subsequent conversions to D- or L-tryptophan, indole-3-pyruvate, MP or monatin. Substituted indoles have been shown in the literature to be suitable substrates for PLP enzymes and have provided substituted tryptophan. Fukuda, D. S., et al., “Production of Substituted L-Tryptophans by Fermentation,” Appl. Environ. Microbiol., 21: 841-43 (1971). Halogen does not appear to sterically hinder the catalytic mechanism of tryptophan synthase beta subunits, and enantiospecificity also remained intact.

In some embodiments, a process is provided for producing a monatin composition which includes producing indole-3-pyruvate from L-tryptophan, producing 2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid ("precursor of monatin "or" MP ") from indole-3-pyruvate and produce monatin from MP. The reaction of L-tryptophan to produce indole-3-pyruvate is facilitated by an enzyme that has higher specificity, greater activity, or both for L-tryptophan than for R-MP, (R, R) -monatin, or both. According to certain embodiments, the indole-3 reaction

Pyruvate is facilitated by an enzyme that has specific aldolase R activity and consequently produces R-MP. In accordance with certain embodiments, a racemase enzyme is provided that can facilitate epimerization of the amino acid derivative product of the tryptophan reaction from an isomeric form to another isomeric form.

In some embodiments, a process is provided for producing a monatin composition which includes producing indole-3-pyruvate from L-tryptophan, producing 2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid ("precursor of monatin "or" MP ") from indole-3-pyruvate and produce monatin from MP. The reaction of L-tryptophan to produce indole-3-pyruvate is facilitated by an enzyme that has higher specificity, greater activity, or both for L-tryptophan than for R-MP, (R, R) -monatin, or both, and the MP's reaction to form monatin is facilitated by an enzyme, which is stereoselective for R-MP. The term "stereoselective" means that one enzyme has greater specificity, greater activity, or both for one isomer, in this case for R-MP than for S-MP, than another. In preferred embodiments, a stereoselective enzyme has limited activity for one isomer compared to another. "Limited" activity means activity that is minimally perceived or not perceived, for example as determined in accordance with experiments provided herein.

It should be noted that, when reference is made to series of reactions such as in the preceding paragraphs, it is not necessary that each stage of the invention be carried out explicitly; it is sufficient that the stages are carried out implicitly. In other words, for example, the process for producing a monatin composition, which includes producing indole-3-pyruvate from L-tryptophan, producing 2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid ( "Monatin precursor" or "MP") from indole-3-pyruvate and producing monatin from MP, where each reaction is facilitated by an appropriate enzyme, can be carried out by combining L-tryptophan with the enzymes and binding conditions such that the listed reactions can occur. In that case, the L-tryptophan can be reacted to produce indole-3-pyruvate, the indole-3-pyruvate produced from the L-tryptophan reaction can be reacted to form MP, and the MP produced from the indole3 reaction -pyruvate can be reacted to form monatin. The process can also be carried out, by way of example, by providing a compound that can produce L-tryptophan, under conditions suitable for the production of L-tryptophan to occur and combining said compound with enzymes capable of facilitating the series of reactions indicated in suitable conditions for such reactions to occur. As another example, the process can be carried out by providing a genetically engineered microorganism to produce monatin according to the described route and by providing appropriate conditions for the fermentation process to occur. For example, a microorganism that naturally produces large amounts of L-tryptophan could be genetically engineered to produce or overproduce one or more of the enzymes used to facilitate monatin pathway reactions, and suitable conditions could be provided so that the microorganism can thus produce monatina.

In other embodiments, a process for producing monatin is provided, in which a substrate forms a Lamino Acid when L-tryptophan is converted to indole-3-pyruvate, indole-3-pyruvate reacts to form MP (which may include R-MP and S-MP although it preferably includes only or predominantly R-MP) and the L-amino acid reacts to regenerate (also called "recycle") the substrate when the R-MP is converted to (R, R) monatin. The reaction of R-MP to form (R, R) -monatin is facilitated by a stereoinverting aminotransferase such as D-methionine aminotransferase (EC 2.6.1.41) or an enzyme that has D-phenylglycine aminotransferase activity.

A process is provided for producing a monatin composition which includes producing D-tryptophan from L-tryptophan, producing indole-3-pyruvate from D-tryptophan, producing R-MP from indole-3-pyruvate and producing (R, R) -monatin from R-MP. The production of D-tryptophan from L-tryptophan is facilitated by a tryptophan racemase and functional equivalents thereof. In certain additional embodiments, the reactions of D-tryptophan to form indole-3-pyruvate and of MP to form monatin are facilitated by the same enzyme. In still other embodiments, the indole-3-pyruvate reaction is facilitated by an enzyme that has specific aldolase R activity and consequently R-MP is formed and the reactions of D-tryptophan to form indole-3-pyruvate and R-MP to form (R, R) -monatin they are facilitated by the same enzyme.

In some embodiments according to the invention there is provided a process for producing a (R, R) -monatin derivative including producing the monatin derivative from a substituted indole-3-pyruvate and pyruvate, using an enzyme having activity of specific aldolase R to catalyze the reaction, as defined in the claims.

Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages in accordance with the invention will be apparent from the description and drawings, and from the claims. As will be appreciated from the description herein, the invention is subject to modifications in different embodiments. Therefore, the drawings and detailed description should be considered as illustrative and not restrictive.

Brief description of the drawings

The following drawings are illustrative of embodiments of the invention and are not intended to limit the scope of the invention, which is defined by the claims.

Figure 1 is a flowchart showing an example of an enzymatic process to produce (R, R) -monatin from L-tryptophan according to the invention. In this example, the process includes using a Laminotransferase (examples of which include an L-tryptophan aminotransferase, a Laromatic aminotransferase, an L-aspartate aminotransferase and an L-alanine aminotransferase) in the reaction of L-tryptophan that has higher specificity and / or selectivity for L-tryptophan as a substrate for R-MP and / or the process includes using an L-amino acid oxidase with specificity and / or limited selectivity for (R, R) -monatin as a substrate. In the specific example diagrammed in Figure 1, an L-aminotransferase or L-amino acid oxidase converts Tryptophan to indole-3-pyruvate, indole-3-pyruvate is reacted with an R-specific aldolase and pyruvate to produce monatin from R -alpha keto acid (R-MP) and R-MP is converted to (R, R) -monatin by a Daminotransferase or a D-amino acid dehydrogenase. As shown in Figure 1, the reactions are reversible but, for the purposes of the invention, the reactions do not need to be carried out in the reverse direction.

Figure 2 is a flow chart showing an example of another process for producing (R, R) -monatin according to the invention. In this example, the process includes using an enzyme to convert R-MP to monatin that is stereoselective for R-MP. The specific example diagrammed in Figure 2 shows the conversion of tryptophan to indole-3-pyruvate in a reverse reaction. Indole-3-pyruvate can be reacted with a non-stereospecific aldolase to reverse form alpha keto acid monatin (R and S-MP). R-MP is inversely converted to (R, R) -monatin by a stereoselective D-aminotransferase or a stereoselective D-amino acid dehydrogenase. Any S-MP that is formed by non-stereospecific aldolase can be converted back to indole-3-pyruvate if a stereoselective D-aminotransferase or D-amino acid dehydrogenase is used. For the purposes of the invention, reactions shown to be reversible need not be carried out in the reverse direction.

Figure 3 is a flowchart showing an example of yet another process to produce (R, R) -monatin from L-tryptophan according to the invention. In this example, the process includes converting L-tryptophan to tryptophan using a tryptophan racemase and using a D-amino acid product in the reaction coupled to the indole-3-pyruvate formation reaction as a substrate in the reaction coupled to the reaction of (R, R) -monatin formation. In the specific example diagrammed in Figure 3, L-tryptophan is converted to D-tryptophan by a tryptophan racemase in a reverse reaction. D-tryptophan is reacted with alpha-ketoglutarate (a-KG) and broadly specific D-aminotransferase to produce indole-3-pyruvate and D-glutamate. Indole-3-pyruvate is reacted with pyruvate and an R-specific aldolase and converted to R-alpha keto acid (R-MP) monatin and R-MP is reacted with broadly specific D-aminotransferase and D-glutamate to form (R, R) -monatin and alpha-ketoglutarate (a-KG). As shown in Figure 3, each of the reactions are reversible but, for the purposes of the invention, it is not necessary that the reactions be carried out in the reverse direction.

Figure 4 is a flowchart showing an example of yet another process to produce (R, R) -monatin from L-tryptophan according to the invention. In this example, the process includes converting the L-amino acid formed in the reaction coupled to the L-tryptophan reaction to a D-amino acid; This D-amino acid acts as an amino donor for the reaction in which R-MP is converted to (R, R) -monatin. In the specific example diagrammed in Figure 4, L-tryptophan is reacted with an L-aminotransferase and alpha-ketoglutarate to produce indole-3-pyruvate and L-glutamate. Indole-3-pyruvate is reacted with pyruvate and a specific aldolase and converted to R-alpha keto acid monatin (R-MP), and R-MP is reacted with a broadly specific Daminotransferase and D-glutamate to form (R, R) -monatin and alpha-ketoglutarate. As shown in Figure 4, the reactions are reversible but, for the purposes of the invention, it is not necessary that the reactions be carried out in the reverse direction.

Figure 5 is a flowchart showing an example of yet another process to produce (R, R) -monatin from L-tryptophan according to the invention. In this example, the process includes enzymatically facilitating the conversion of R-MP to (R, R) -monatin using a stereoinverting enzyme such that the L-amino acid formed by the reaction coupled to the L-tryptophan reaction can be used as substrate for the reaction coupled to the reaction of R-MP with (R, R) -monatin. In the specific example diagrammed in Figure 5, L-tryptophan is reacted with an L-aminotransferase and oxaloacetate, pyruvate or alpha-ketoglutarate (a-KG) to produce indole-3-pyruvate and L-aspartate (if oxaloacetate is used) , L-alanine (if pyruvate is used) or L-glutamate (if a-KG is used). Indole-3-pyruvate is reacted with pyruvate and an R-specific aldolase and converted to R-alpha keto acid (R-MP) monatin, and R-MP is reacted with a stereoinverting aminotransferase and L-aspartate, Lalanin or L-glutamate to form (R, R) -monatin and oxaloacetate (if L-aspartate is used), pyruvate (if Lalanine is used) or alpha-ketoglutarate (a-KG, if L-glutamate is used). As shown in Figure 5, the reactions are reversible but, for the purposes of the invention, the reactions need not be carried out in the reverse direction.

Figure 6 is a flowchart showing an example of yet another process to produce (R, R) -monatin according to the present invention. In this example, the process includes recycling the L-amino acid produced in the indole-3-pyruvate formation reaction with the D-amino acid used as a reagent with R-MP in the (R, R) -monatin formation reaction by a series of conversion reactions. In the specific example diagrammed in Figure 6, L-tryptophan is reversibly reacted with an L-aminotransferase and oxaloacetate to produce indole-3-pyruvate and L-aspartate. Indole-3-pyruvate is reversibly reacted with pyruvate and an R-specific aldolase and converted to R-alpha keto acid monatin (R-MP) and R-MP is reversibly reacted with a D-aminotransferase and D- Alanine to form (R, R) -monatin and pyruvate. L-aspartate is converted to L-alanine and CO2 using a 4-decarboxylase aspartate. L-Alanine is converted to D-Alanine with an alanine racemase. For the purposes of the invention, reactions shown to be reversible need not be carried out in the reverse direction.

Figure 7 is a flowchart showing an example of yet another process to produce (R, R) -monatin according to the present invention. In this example, the process includes advancing the L-tryptophan reaction (i.e., boosting the reaction towards the production of indole-3-pyruvate) by converting the L-amino acid derivative product of said reaction into another product. In this example, the L-aspartate derivative product of Lamino Acid is converted to L-alanine in an irreversible reaction using a decarboxylase. In the specific example diagrammed in Figure 7, L-tryptophan is reacted reversibly with an L-aminotransferase and with alpha-ketoglutarate (a-KG) or oxaloacetate to produce indole-3-pyruvate and L-glutamate (if using a- KG) or Laspartate (if oxaloacetate is used). Indole-3-pyruvate is reacted reversibly with pyruvate and an R-specific aldolase and converted to R-alpha keto acid monatin (R-MP). R-MP is reversibly reacted with a D-aminotransferase and a D-amino acid to form (R, R) -monatin and either oxaloacetate, pyruvate or a-KG. The L-glutamate or L-aspartate that was produced by the Laminotransferase reaction is converted to 4-aminobutanoate and CO2 (if the substrate is L-glutamate) or to �-alanine and CO2 (if the substrate is L-aspartate) using a glutamic acid or an aspartate decarboxylase. For the purposes of the invention, reactions shown to be reversible need not be carried out in the reverse direction.

Figure 8 is a flowchart showing an example of yet another process to produce (R, R) -monatin according to the present invention. In this example, the process includes recycling the amino acid derivative product of the L-tryptophan reaction with the amino acid reagent from the R-MP reaction by a series of conversion reactions. In the specific example diagrammed in Figure 8, L-tryptophan is reacted reversibly with an L-aminotransferase and with alpha-ketoglutarate (a-KG) to produce indole-3-pyruvate and Lglutamate. Indole-3-pyruvate is reacted reversibly with pyruvate and an R-specific aldolase and converted to R-alpha keto acid monatin (R-MP). R-MP is reversibly reacted with a Daminotransferase and D-alanine to form (R, R) -monatin and pyruvate. An L-alanine aminotransferase and pyruvate are used to reversibly convert the L-glutamate that was a product of the reaction of the L-aminotransferase back to a-KG, with L-alanine as a co-product. An alanine racemase reversibly converts Lalanine to D-alanine which is useful in the third reaction (the D-aminotransferase reaction). For the purposes of the invention, reactions shown to be reversible need not be carried out in the reverse direction.

Figure 9 block diagram of a computer system.

Figure 10 is a flow chart illustrating an embodiment of a process for comparing a new protein or nucleotide sequence to a sequence database to determine levels of homology between the new sequence and the sequences in the database.

Figure 11 is a flow chart illustrating an embodiment of a process on a computer to determine if two sequences are homologous.

FIG. 12 is a flow chart illustrating an embodiment of an identification process 300 for detecting the presence of a feature in a sequence.

Figures 13 and 14 together illustrate the activities of 58 different aldolases (each identified by their specific SEQ ID number) in monatin precursor (MP) formation as measured by LC / MS / MS.

Figure 15 illustrates the effect of dithiothreitol on monatin production by the aldolase-active polypeptide of SEQ ID NO: 88.

Similar reference symbols in the various drawings indicate similar elements.

Detailed description

Various embodiments have been previously described and will be described in more detail below. Embodiments of the invention include one or more of the described aspects.

Abbreviations and terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those skilled in the art in practicing the present disclosure. As used herein, "including" means "comprising". Furthermore, the singular forms "a", "an" or "the" or "the" include references to the plural, unless the context clearly indicates otherwise. For example, the reference to "comprising a protein" includes one or more of said proteins, and the reference to "comprising cells" includes the reference to one or more cells and equivalents thereof known to those skilled in the art and so on. The term "approximately" encompasses the range of experimental error present in any measurement. Unless otherwise indicated, all measurement numbers are assumed to have the word "approximately" in front of them, even if the word "approximately" is not expressly used.

Conservative substitution: a substitution of one amino acid for another amino acid in a polypeptide, which has little to no impact on the activity of the polypeptide. The substitution is considered conservative regardless of whether the exchanged amino acids have a similar structural or functional appearance. For example, ideally, a tryptophan aminotransferase polypeptide that includes one or more conservative substitutions retains tryptophan aminotransferase activity. A polypeptide may be produced to contain one or more conservative substitutions by manipulation of the nucleotide sequence encoding this polypeptide using, for example, conventional procedures such as site-directed mutagenesis or PCR or other methods known in the art.

Non-exhaustive examples of amino acids that can be replaced by an original amino acid in a protein and that are considered conservative substitutions if there is little to no impact on the activity of the polypeptide include: Ala substituted by ser or thr; arg replaced by gln, his or lys; asn replaced by glu, gln, lys, his, asp; asp substituted by asn, glu or gln; cys replaced by ser or ala; gln replaced by asn, glu, lys, his, asp or arg; glu substituted by asn, gln lys or asp; gly replaced by pro; his replaced by asn, lys, gln, arg, tyr; ile replaced by leu, met, val, phe; leu replaced by ile, met, val, phe; lys replaced by asn, glu, gln, his, arg; met replaced by ile, leu, val, phe; phe substituted by trp, tyr, met, ile or leu; be replaced by thr, ala; thr replaced by being or wing; trp replaced by phe, tyr; tyr replaced by his, phe or trp; and val replaced by met, ile, leu.

Additional information about conservative substitutions can be found in, among other places, Ben-Bassat et al., (J. Bacteriol. 169: 751-7, 1987), O'Regan et al., (Gene 77: 237-51, 1989), Sahin-Toth et al., (Protein Sci. 3: 2407, 1994), Hochuli et al., (Bio / Technology 6: 1321-5, 1988), WO 00/67796 (Curd et al. ) and in conventional genetics and molecular biology textbooks.

Derived: For the purposes of the specification and claims, a substance is "derived" from an organism or source if one or more of the following statements is true: 1) the substance is present in the organism / source; 2) the substance is removed from the native host; or 3) the substance is removed from the native host and evolves, for example, by mutagenesis.

Isolated: The term "isolated", as used herein, refers to any substance that is removed from its native host; the substance does not need to be purified. For example "isolated nucleic acid" refers to a natural nucleic acid that is not immediately contiguous to both sequences to which it is immediately contiguous (one at the 5 'end and one at the 3' end) in the natural genome of the organism of the which derives. For example, an isolated nucleic acid may be, but is not limited to, a recombinant DNA molecule of any length, provided that one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a natural genome is removed or is absent. Thus, an isolated nucleic acid includes, but is not limited to, a recombinant DNA that exists as a separate molecule (such as a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences, as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (eg, a retrovirus, adenovirus, or herpetic virus) or into the genomic DNA of a prokaryote or eukaryote. Furthermore, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

As used herein, the term "isolated" means that the material (such as a protein or nucleic acid according to the invention) is removed from its original environment (such as the natural environment if it is natural). For example, a natural polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Said polynucleotides can be part of a vector and / or said polynucleotides or polypeptides can be part of a composition and still be isolated, since said vector or composition is not part of its natural environment.

The term "isolated", as used herein in relation to nucleic acid, also includes any artificial nucleic acid, since artificial nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a natural genome. . For example, artificial nucleic acid such as a

Genetically manipulated nucleic acid is considered to be an isolated nucleic acid. Genetically engineered nucleic acid can be obtained using common molecular cloning techniques or chemical nucleic acid synthesis. The isolated artificial nucleic acid can be independent of other sequences or incorporated into a vector, an autonomously replicating plasmid, a virus (eg, a retrovirus, adenovirus, or herpetic virus), or the genomic DNA of a prokaryote or eukaryote. Furthermore, an artificial nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

Purified: The term "purified" as used herein does not require absolute purity, but is intended to be a relative term. Thus, for example, a purified polypeptide or nucleic acid preparation may be one in which the polypeptide or nucleic acid in question is in a higher concentration than the polypeptide or nucleic acid would be in its natural environment within an organism or a concentration greater than that of the environment from which it was withdrawn.

Individual nucleic acids obtained from a library have been conventionally purified to achieve electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly from the library or from total human DNA. The purified nucleic acids disclosed herein were purified from the rest of the genomic DNA in the body at least 104-106 times. In some embodiments, the term "purified" includes nucleic acids that were purified from the rest of the genomic DNA or from other sequences in a library or other environment by at least an order of magnitude, such as, in some embodiments, two or more orders or four or five orders of magnitude.

Amino Acid: "Amino acid" or "amino acid sequence", as used herein, refers to a sequence of oligopeptides, peptides, polypeptides or proteins or to a fragment, portion or subunit of any of these, and to natural or synthetic molecules . "Amino acid" or "amino acid sequence" includes a sequence of oligopeptides, peptides, polypeptides or proteins or a fragment, portion or subunit of any of these, and natural or synthetic molecules. The term "polypeptide", as used in the present, refers to amino acids linked together by peptide bonds or modified peptide bonds, ie, peptide isosters, and may contain modified amino acids other than the 20 gene-encoded amino acids. Polypeptides can be modified by natural processes, such as post processing -translational or by chemical modification techniques well known in the art Modifications can occur anywhere in the polypeptide, including the central structure of the peptide, the amino acid side chains and the amino or carboxyl termini. type of modification may be present to the same degree or to different degrees at various sites in a polypeptide dice. Also, a given polypeptide can have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, crosslinking cyclization, disulfide bond formation, demethylation, covalent intermolecular bond formation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristollation, oxidation, pegylation, glucan hydrolase processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and protein transfer RNA mediated addition of amino acids such as arginylation. (See Creighton, TE, Proteins - Structure and Molecular Properties 2nd Ed., WH Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)). The peptides and polypeptides disclosed herein also include all "mimetic" and "peptidomimetic" forms, as described in greater detail below.

Polypeptide having aldolase activity: "Polypeptide having aldolase activity" means a polypeptide which alone or in association with one or more additional polypeptides (having the same or a different sequence) is a protein with the enzymatic activity of an aldolase.

Recombinant: "Recombinant" polypeptides or proteins refers to polypeptides or proteins produced by recombinant DNA techniques; that is, produced from cells transformed by an exogenous DNA construct that encodes the desired polypeptide or protein. Polypeptides or "synthetic" proteins are those prepared by chemical synthesis. Chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments disclosed herein. Such a method has been known in the art since the early 1960s (Merrifield, R. B., J. Am. Chem. Soc., 85: 2149-2154, 1963) (See also Stewart, J.

M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., Pgs. 11-12)) and were recently used in commercially available laboratory design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally used the findings of H. M. Geysen et al., Proc. Natl. Acad. Sci., USA, 81: 3998 (1984) and provide synthesizing peptides on the tips of multiple "rods" or "pins", all of which are connected to a single plate.

Basically identical: The phrase "basically identical", in the context of two nucleic acids or polypeptides, refers to two or more sequences that have, for example, at least about 50%, 51%, 52%, 53%, 54% , 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%,

75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (sequence) identity of nucleotide or amino acid residues when compared and aligned for maximum correspondence, based on it is measured using one of the known sequence comparison algorithms or by visual inspection. In other embodiments, the basic identity exists in a region of at least about 100 or more residues, and more commonly the sequences are basically identical at least about 150 to 200 or more residues. In some embodiments, the sequences are basically identical over the full length of the coding regions.

Furthermore, a "basically identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions. In some embodiments, the substitution occurs at a site that is not the active site of the molecule or, alternatively, the substitution occurs at a site that is the active site of the molecule, provided that the polypeptide essentially retains its functional (enzymatic) properties. . A conservative amino acid substitution, for example, substitutes one amino acid for another of the same kind (such as substituting one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substituting one polar amino acid for another , such as the substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids, for example, can be removed from an aldolase polypeptide, such as pyruvate aldolase, HMG and / or KHG aldolase, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino or carboxyl-terminal amino acids that are not necessary for the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be removed. The modified polypeptide sequences disclosed herein can be evaluated for biological activity of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, by various methods, including contacting the modified polypeptide sequence with a substrate and determining whether the modified polypeptide decreases the amount of specific substrate in the assay or increases the bioproducts of the enzymatic reaction of an aldolase functional polypeptide, such as pyruvate aldolase, such as HMG and / or KHG aldolase with the substrate.

Fragment: A "fragment", as used herein with respect to a protein or polypeptide or nucleic acid, is a portion of the protein, polypeptide or nucleic acid, respectively. The fragments can have the same or basically the same amino acid or nucleic acid sequence as the longest protein, polypeptide or nucleic acid sequence from which the fragment is derived. Fragments having different three-dimensional structures compared to those of longer proteins, polypeptides, or nucleic acids are also included. An example of this is a "pro-forma" molecule, such as a low-activity proprotein that can be modified by cleavage to produce a mature enzyme with considerably higher activity. A fragment of a protein or polypeptide can be an enzymatically active portion of a protein or polypeptide.

Stereoinverting Aminotransferase: A "stereoinverting aminotransferase" is a polypeptide capable of preferentially or selectively producing a chiral amino acid product (such as monatin) while using a substrate of opposite chirality as an amino donor. For example, a stereoinverting aminotransferase may be a D-phenylglycine aminotransferase (also called D-4-hydroxyphenylglycine aminotransferase) that preferably or selectively uses L-glutamate as a substrate to produce (R, R) -monatin. Non-exhaustive examples of stereoinverting aminotransferases include D-methionine aminotransferase (EC 2.6.1.41) and enzymes that have D-phenylglycine aminotransferase activity or D-4-hydroxyphenylglycine aminotransferase activity.

The disclosure provides aldolase-active polypeptides, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, polynucleotides encoding them, and methods of preparing and using these polynucleotides and polypeptides. In some embodiments, the disclosure also provides aldolase enzymes, such as pyruvate aldolase enzymes, such as HMG and / or KHG aldolase, polynucleotides encoding these enzymes, the use of such polynucleotides, and polypeptides.

The disclosure provides aldolases, such as pyruvate aldolases, HMG and / or KHG aldolases, modified or evolved with a higher specific activity compared to unmodified or evolved aldolases, respectively.

Aldolases, such as a pyruvate aldolase, such as, but not limited to, an HMG and / or a KHG aldolase, are provided which facilitate the production of a 3,4-substituted 2-keto-glutarate. The disclosure provides a method of preparing a 3,4-substituted 2-keto glutarate comprising: (a) providing a polypeptide having aldolase activity, such as pyruvate aldolase activity, such as, non-limitingly, an HMG aldolase and / or KMG aldolase activity; (b) providing a donor and an acceptor compound; and (c) contacting the polypeptide of step (a) with the compounds of step (b) under conditions in which aldolase catalyzes the synthesis of a 3,4-substituted 2-keto-glutarate, where optionally the donor and acceptor are a pyruvate or a pyruvate donor and an a-keto acid acceptor, a ketone and / or an aldehyde.

A pyruvate aldolase, such as an HMG and / or a KHG aldolase, can be used in conjunction with a D-aminotransferase to prepare a 4-substituted D-glutamic acid or a derivative thereof. A 4-substituted D-glutamic acid and / or a derivative thereof can be used as an antibiotic, since these compounds have been found to inhibit

bacterial glutamate racemase. The disclosure provides a method of preparing a 4-substituted D-glutamic acid comprising: (a) providing a polypeptide having an aldolase activity, such as a pyruvate aldolase activity, such as, non-exhaustively, an HMG aldolase activity and / or KMG aldolase; (b) providing an a-keto acid acceptor and a pyruvate or a pyruvate donor; and (c) contacting the polypeptide of step (a) with the compounds of step (b) under conditions in which aldolase catalyzes the synthesis of a 4-substituted D-glutamic acid, where optionally the polypeptide has pyruvate aldolase, HMG aldolase and / or KHG aldolase activity and optionally the method further comprises the use of a Daminotransferase.

The disclosure provides compositions (such as enzyme preparations, food and food additives, feed and feed additives, beverages and beverage additives, drugs and drug additives, and food supplements) comprising the enzymes, polypeptides or polynucleotides as disclosed at the moment. These compositions can be formulated in a variety of forms, such as liquids, gels, pills, tablets, sprays, films, micelles, powders, food, feed granules, or encapsulated forms, including nanoencapsulated forms.

Assays for measuring aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity, such as for determining whether a polypeptide has aldolase activity, including pyruvate activity such as, Non-limiting, HMG and / or KHG aldolase activity are well known in the art; see E.E. Dekker & R.P. Kitson, J. Biol. Chem. 267, 10507-10514, 1992; Taha T S, Deits TL, Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage, Biochem Biophys Res Commun. 1994 Apr. 15; 200 (1): 459-66; Dekker EE, Kobes RD, Grady SR, 2-keto-4-hydroxyglutarate aldolase from bovine liver, Methods Enzymol. 1975; 42: 280-5; Dekker EE, Nishihara H, Grady SR, Methods Enzymol. 1975; 42: 285-90, 2-keto-4hydroxyglutarate aldolase from Escherichia coli; Nishihara H, Dekker EE, Biochim Biophys Acta. 1969 Jul 8; 185 (1): 255-7, A stereospecific 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. An example of a suitable assay to determine if a polypeptide has aldolase activity, such as pyruvate aldolase activity, such as HMG and / or KHG aldolase is described in Example 3.

Aldolases can be used effectively with a variety of pH conditions, including, for example, a range from about 3.0 to about 12.0. Aldolases can be used at approximately pH 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 , 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 or about 12.0. Reaction conditions carried out under acidic or alkaline conditions, such as in certain industrial or pharmaceutical applications of enzymes disclosed herein, may also be advantageous.

The disclosure provides aldolase polypeptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase as disclosed herein in a variety of forms and formulations. Aldolase polypeptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase as disclosed herein in a variety of forms and formulations are used in the methods disclosed herein. For example, purified aldolase polypeptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be used in enzyme preparations deployed in the production of R-2-hydroxy 2- (indole-3-ylmethyl) -4- acid. ketoglutaric (R-MP) and certain monatin stereoisomers, such as (R, R) - and (S, R) -monatin and salts thereof, as well as certain stereoisomers of monatin derivatives, such as derivative configurations of (R, R) - and (S, R) -monatin and salts thereof or pharmaceutical or food aid applications. Alternatively, the enzymes disclosed herein can be used directly in processes to produce R-2-hydroxy 2- (indole-3-methylmethyl) -4-ketoglutaric acid (R-MP) and certain monatin stereoisomers, such as (R, R) -monatin and salts thereof, as well as certain stereoisomers of monatin derivatives, such as configurations of (R, R) -monatin derivatives and salts thereof, for processing food, liquids, feed and the like.

In some embodiments, aldolase polypeptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein can be expressed in a microorganism using procedures known in the art. The aldolase polypeptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein can be immobilized on a solid support prior to use in the methods disclosed herein. Methods for immobilizing enzymes on solid supports are commonly known in the art, for example J. Mol. Cat. B: Enzymatic 6 (1999) 29-39; Chivata et al. Biocatalysis: Immobilized cells and enzymes, J. Mol. Cat. 37 (1986) 1-24: Sharma et al., Immobilized Biomaterials Techniques and Applications, Angew. Chem. Int. Ed. Engl. 21 (1982) 837-54: Laskin (Ed.), Enzymes and Immobilized Cells in Biotechnology.

Nucleic acids, probes, and inhibitory molecules

The disclosure provides isolated and recombinant nucleic acids such as those in the Sequence Listing; Nucleic acids encoding polypeptides, including the polynucleotide sequences disclosed herein, such as those in the Sequence Listing; including expression cassettes such as expression vectors and various cloning vehicles comprising nucleic acids disclosed herein. In some embodiments, the disclosure also includes methods for discovering, identifying, or isolating aldolase polypeptide sequences, such as new pyruvate aldolase, HMG, and / or KHG aldolase using the nucleic acids disclosed in

the present. The disclosure also includes methods of inhibiting the expression of aldolase-encoding genes and transcripts, such as pyruvate aldolase, such as HMG and / or KHG aldolase using the nucleic acids disclosed herein.

Additionally methods are provided for modifying the nucleic acids disclosed herein, including making nucleic acid variants disclosed herein by, for example, synthetic ligation reassembly, optimized targeted evolution system, and / or saturation mutagenesis such as gene site saturation (GSSM). The term "saturation mutagenesis", Gene Site Saturation Mutagenesis, or "GSSM" includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail below. The term "optimized targeted evolution system" or "optimized targeted evolution" includes a method for reassembling fragments of related nucleic acid sequences, such as related genes, and is explained in detail below. The term "synthetic ligation reassembly" or "SLR" includes a method for ligating oligonucleotide fragments non-stochastically and is explained in detail below. The term "variant" refers to polynucleotides or polypeptides disclosed herein modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) that still retain the biological activity of an aldolase, such as pyruvate aldolase , HMG and / or KHG aldolase disclosed herein. Variants can be produced by various means including methods such as, for example, error-prone PCR, transposition, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, ensemble recursive mutagenesis, mutagenesis ensemble exponential, site specific mutagenesis, gene reassembly, GSSM, and combinations thereof.

The nucleic acids disclosed herein can be made, isolated and / or managed by, for example, cloning and expression of cDNA libraries, amplification of messenger or genomic DNA by PCR, and the like. For example, the sequences disclosed herein were initially derived from environmental sources. Accordingly, the disclosure provides aldolase-encoding nucleic acids, such as pyruvate aldolase, such as the HMG and / or KHG aldolase enzyme and the polypeptides encoded therein, preferably derived from a common source, such as an environmental, mixed culture, or a bacterial source.

By practicing the methods disclosed herein, homologous genes can be modified using a template nucleic acid, as described herein. The methods can be practiced in conjunction with any method or protocol or device known in the art, which have been described in the scientific and patent literature.

The phrases "nucleic acid" or "nucleic acid sequence", as used herein, refer to an oligonucleotide, nucleotide, polynucleotide or a fragment of any of these, DNA or RNA of genomic or synthetic origin, the which can be single-stranded or double-stranded and can represent a sense or antisense (complementary) chain with respect to peptide nucleic acid (PNA) or any material similar to DNA or similar to RNA of natural or synthetic origin. The phrases "nucleic acid" or "nucleic acid sequence" include oligonucleotides, nucleotides, polynucleotides or a fragment of any of these, DNA or RNA (such as mRNA, rRNA, tRNA, RNAi) of genomic or synthetic origin, which they can be single-stranded or double-stranded and can represent a sense or antisense chain with respect to peptide nucleic acid (PNA)

or to any DNA-like or RNA-like material of natural or synthetic origin, including, for example, RNAi, ribonucleoproteins (such as double-stranded RNAi, such as RNPi). The term encompasses nucleic acids, that is, oligonucleotides containing known analogs of natural nucleotides. The term further encompasses nucleic acid-like structures with synthetic backbones, see for example Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197; Strauss-Soukup (1997) Biochemistry 36: 8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156. "Oligonucleotide" includes a single-chain polydeoxynucleotide or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides have no 5 'phosphate and therefore will not bind to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can bind to a fragment that has not been dephosphorylated.

A "coding sequence for" or a "nucleotide sequence encoding" a specific polypeptide or protein, is a nucleic acid sequence that can be transcribed and translated to a polypeptide or protein when placed under the control of appropriate regulatory sequences. The term "gene" means the DNA segment that participates in the production of a polypeptide chain; it includes regions preceding and following the coding region (leader and back) as well as, where appropriate, intercalated sequences (introns) between individual coding segments (exons). A promoter sequence is "operably linked" to a coding sequence when the RNA polymerase that initiates transcription in the promoter will transcribe the coding sequence to mRNA. "Operably linked" as used herein refers to a relationship between two or more nucleic acid segments (such as DNA). It can refer to the functional relationship of the transcriptional regulatory sequence to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a nucleic acid disclosed herein, if it stimulates or modulates transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence

they are physically contiguous to the transcribed sequence, that is, they act in cis. However, some transcriptional regulatory sequences, such as enhancers, must not be physically contiguous or located in close proximity to the coding sequences, the transcription of which they enhance.

The term "expression cassette" as used herein refers to a nucleotide sequence that is capable of affecting the expression of a structural gene (ie, a protein coding sequence, such as an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase according to the invention) in a host compatible with said sequences. Expression cassettes include at least one promoter operably linked to the polypeptide coding sequence; and, optionally, with other sequences, such as transcription termination signals. Additional necessary or useful factors may also be used to produce expression, such as enhancers, alpha factors. Accordingly, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a bare nucleic acid.

or a nucleic acid bound to a protein or lipid. The vector optionally comprises nucleic acids and / or proteins and / or viral or bacterial membranes (such as a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (such as RNA replicons, bacteriophages) to which DNA fragments can bind and replicate. Accordingly, vectors include, but are not limited to, circular or linear self-replicating autonomous RNA, DNA, or RNA (such as plasmids, viruses, and the like, see US Patent No. 5,217,879) and include expression plasmids and do not expression. When a recombinant microorganism or cell culture is described as a host of an "expression vector", this includes circular and linear extrachromosomal DNA and DNA that has been incorporated into the host chromosome (s). When a vector is maintained in a host cell, the vector can stably replicate through the cells during mitosis as an autonomous structure or is incorporated into the host genome.

As used herein, the term "recombinant" encompasses nucleic acids adjacent to a "main chain" nucleic acid to which they are not adjacent in their natural environment. In some embodiments, to "enrich" the nucleic acids will represent approximately 5% or more of the nucleic acid inserts in a population of nucleic acid backbone molecules. Backbone molecules include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or handle a nucleic acid insert of interest. In some embodiments, the enriched nucleic acids represent approximately 15% or more of the nucleic acid inserts in the population of recombinant backbone molecules. In some embodiments, the enriched nucleic acids represent approximately 50% or more of the nucleic acid inserts in the population of recombinant backbone molecules. In some embodiments, the enriched nucleic acids represent approximately 90% or more of the nucleic acid inserts in the population of recombinant backbone molecules.

There is disclosed an isolated synthetic or recombinant nucleic acid comprising one of the sequences according to the invention, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 or more consecutive bases of a nucleic acid disclosed herein. Isolated synthetic or recombinant nucleic acids can comprise DNA, including cDNA, genomic DNA, and synthetic DNA. DNA can be double-stranded or single-stranded and if it is single-stranded it can be the coding strand or the non-coding (antisense) strand. Alternatively, the isolated synthetic or recombinant nucleic acids comprise RNA.

The isolated synthetic or recombinant nucleic acids disclosed herein can be used to prepare one of the disclosed polypeptides or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more consecutive amino acids from one of the polypeptides disclosed herein. Accordingly, an isolated recombinant or synthetic nucleic acid encoding one of the polypeptides disclosed herein or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 is also disclosed. or 150 or more consecutive amino acids from one of the polypeptides disclosed herein. The coding sequences of these nucleic acids may be identical to one of the coding sequences of one of the nucleic acids disclosed herein or may be different coding sequences having at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more consecutive amino acids of one of the polypeptides disclosed herein, as a result of redundancy or degeneracy of the genetic code. The genetic code is known to those skilled in the art and can be obtained for example on page 214 of B. Lewin, Genes VI, Oxford University Press, 1997.

The isolated nucleic acid encoding one of the polypeptides disclosed herein and sequences substantially identical thereto, may include, but are not limited to: the coding sequence for a nucleic acid disclosed herein and additional coding sequences, such as leader sequences or proprotein sequences and non-coding sequences, such as introns or non-coding sequences 5 'and / or 3' of the coding sequence. Therefore, as used herein, the term "polynucleotide that encodes

a "polypeptide" encompasses a polynucleotide that includes the coding sequence for the polypeptide, as well as a polynucleotide that includes additional coding and / or non-coding sequence.

Alternatively, the nucleic acid sequences disclosed herein and sequences substantially identical thereto, can be mutaged using conventional techniques, such as site-directed mutagenesis or other techniques known to those skilled in the art, to introduce silent changes to the polynucleotides disclosed in the present. As used herein, "silent changes" include, for example, changes that do not alternate the amino acid sequence encoded by the polynucleotide. Such changes may be desirable to increase the level of the polypeptide produced by host cells that contain a vector encoding the polypeptide by introducing codons or codon pairs that frequently occur in the host organism.

The disclosure also relates to polynucleotides that have nucleotide changes that result in amino acid substitutions, additions, deletions, fusions, and truncation in the polypeptides disclosed herein. Such nucleotide changes can be introduced using techniques such as site-directed mutagenesis, random chemical mutagenesis, exonuclease III deletion, and other recombinant DNA techniques. Alternatively, such nucleotide changes may be naturally occurring allelic variants that are isolated by identifying nucleic acids that specifically hybridize to probes comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75 , 100, 150, 200, 300, 400 or 500 consecutive bases of one of the sequences disclosed herein (or sequences complementary thereto) under conditions of high, moderate or low stringency as provided herein.

General techniques

The nucleic acids used to implement this method, be they RNA, siRNA, miRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically manipulated, recombinantly amplified and / or expressed / generated. Recombinant polypeptides (such as aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase) generated from these nucleic acids can be isolated or cloned and individually evaluated to determine if they have a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro by known chemical synthesis techniques, such as those described, for example, in Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; United States Patent No. 4,458,066.

Techniques for nucleic acid management, such as subcloning, labeling probes (such as random primer labeling using Klenow polymerase, notch translation, amplification), sequencing, hybridization, and the like are described in the scientific and patent literature, see Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Volumes 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and handling nucleic acids used to practice the methods disclosed herein is to clone from genomic samples and, if desired, to screen and re-clone inserts isolated or amplified from, for example. , genomic clones or cDNA clones. The nucleic acid sources used in the methods disclosed herein include those contained in genomic or cDNA libraries, such as mammalian artificial chromosomes (MACs), see US Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see Rosenfeld (1997) Nat. Genet. 15: 333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see Woon (1998) Genomics 50: 306-316; P1-derived vectors (PACs), see Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages, or plasmids.

A nucleic acid encoding a polypeptide disclosed herein is assembled at the appropriate stage with a leader sequence capable of directing secretion of the translated polypeptide or a fragment thereof.

The disclosure provides fusion proteins and nucleic acids that encode them. A polypeptide disclosed herein can be fused with a heterologous peptide or polypeptide, such as N-terminus identification peptides that impart desired characteristics, such as increased stability or simplified purification. The peptides and polypeptides disclosed herein can also be synthesized and expressed as fusion proteins with one or more additional domains attached thereto, for example, to produce a more immunogenic peptide, to more easily isolate a recombinantly synthesized peptide, to identify and isolating antibodies and B cells expressing antibodies and the like. The domains that facilitate detection and purification

They include, for example, metal chelation peptides such as polyhistidine tracts and histidine tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the purification system by extension / affinity FLAGS (Immunex Corp, Seattle Wash.). Inclusion of cleavable sequences by a linker such as Factor Xa or enterokinase (Invitrogen, Carlsbad, CA) between a purification domain and the peptide or polypeptide comprising the motif to facilitate purification. For example, an expression vector may include a nucleic acid sequence encoding an epitope bound to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see for example Williams (1995) Biochemistry 34: 1787-1797; Dobeli (1998) Protein Expr. Purif. 12: 404-414). Histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means to purify the epitope from the rest of the fusion protein. The technology related to vectors encoding fusion proteins and the application of fusion proteins is described in the scientific and patent literature, see for example Kroll (1993) DNA Cell. Biol., 12: 441-53.

Transcription and translation control sequences

The disclosure provides nucleic acid sequences (such as DNA) disclosed herein operably linked to expression control sequence (s) (such as transcription or translation), such as promoters or enhancers, to direct or modulate synthesis / expression RNA. The expression control sequence may be in an expression vector. Exemplary bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Exemplary eukaryotic promoters include the immediate-early CMV, the thymidine kinase from HSV, the early and late SV40, LTR from retroviruses, and mouse metallothionein I.

As used herein, the term "promoter" includes all sequences capable of activating transcription of a coding sequence in a cell, such as a plant or animal cell. Accordingly, promoters used in the constructs disclosed herein include cis-acting transcription control elements and regulatory sequences that participate in the regulation or modulation of the transcription time and / or rate of a gene. For example, a promoter can be a cis-acting transcription control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5 'and 3' untranslated regions or an intronic sequence, involved in the regulation of transcription. These cis-acting sequences can interact with proteins or other biomolecules to carry out (activate / deactivate, regulate, modulate, etc.) transcription. "Constitutive" promoters are those that activate expression continuously in most environmental conditions and states of cell development or differentiation. The "inducible" or "regulatable" promoters direct the expression of the nucleic acid disclosed herein under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that can affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.

"Tissue specific" promoters are transcription control elements that are active only in specific cells or tissues or organs, such as in plants or animals. Tissue specific regulation can be achieved by certain intrinsic factors that ensure the expression of genes that encode specific proteins for a given tissue. Such factors are known to exist in mammals and plants to allow the development of specific tissues.

Suitable promoters for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lacI promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons that encode glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter. Eukaryotic promoters include the CMV immediate-early promoter, the HSV thymidine kinase promoter, heat shock promoters, the SV40 early and late promoter, retrovirus LTR, and the mouse metallothionein-I promoter. Other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses can also be used. Suitable promoters to express the polypeptide or fragment thereof in bacteria include the E. coli lac or trp promoters, the lacI promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter. Fungal promoters include the factor a promoter. Eukaryotic promoters include the CMV immediate-early promoter, the HSV thymidine kinase promoter, heat shock promoters, the SV40 early and late promoter, retrovirus LTR, and the mouse metallothionein-I promoter. Other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses can also be used.

Tissue Specific Plant Promoters

The disclosure provides expression cassettes that can be tissue-specific expressed, such as these can express an aldolase, such as pyruvate aldolase, HMG enzyme and / or KHG aldolase disclosed herein in a tissue specific manner. The disclosure also provides plants or seeds that express an aldolase, such as pyruvate aldolase, HMG enzyme and / or KHG aldolase specifically disclosed herein.

of tissue. Tissue specificity can be seed specific, stem specific, leaf specific, root specific, fruit specific, and the like.

The term "plant" includes whole plants, plant parts (such as leaves, stems, flowers, roots, etc.), plant protoplasts, seed cells, and plants and progeny thereof. The plant class that can be used in the method disclosed herein is generally as broad as the class of higher plants susceptible to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including the polyploid, diploid, haploid, and hemizygous states. As used herein, the term "transgenic plant" includes plants or plant cells into which a heterologous nucleic acid sequence has been inserted, such as nucleic acids and various recombinant constructs (such as expression cassettes) disclosed in the present.

In some embodiments, a constitutive promoter such as the CaMV 35S promoter can be used for expression in specific parts of the plant or seed or throughout the plant. For example, for overexpression, a plant promoter fragment can be employed that will direct the expression of a nucleic acid in some or all of the tissues of a plant, such as a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and stages of cell development or differentiation. Examples of constitutive promoters include the 35S cauliflower mosaic virus (CaMV) transcription initiation region, the 1 'or 2' promoter derived from Agrobacterium tumefaciens T-DNA, and other transcription initiation regions of various known plant genes. for experts. Such genes include, for example, ACT11 from Arabidopsis (Huang (1996) Plant Mol. Biol. 33: 125-139); Arabidopsis Cat3 (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet. 251: 196-203); the gene encoding the stearoyl acyl desaturase carrier protein from Brassica napus (Genbank No. X74782, Solocombe (1994) Plant Physiol. 104: 1167-1176); Corn GPc1 (GenBank No. X15596; Martinez (1989) J. Mol. Biol. 208: 551-565); corn Gpc2 (GenBank No. U45855, Manjunath (1997) Plant Mol. Biol. 33: 97-112); plant promoters described in United States Patent Nos. 4,962,028; 5,633,440.

The disclosure uses tissue-derived or constitutive promoters derived from viruses that may include, for example, the subgenomic promoter of tobamovirus (Kumagai (1995) Proc. Natl. Acad. Sci. United States 92: 16791683; the rice tungro bacilliform virus ( RTBV), which replicates only in phloem cells of infected rice plants, with its promoter that activates the intense expression of the phloem-specific reporter gene; the promoter of cassava rib mosaic virus (CVMV), with higher activity in vascular elements, in leaf mesophyll cells and in root tips (Verdaguer (1996) Plant Mol. Biol. 31: 1129-1139).

In some embodiments, the plant promoter directs expression of the nucleo acid expressing the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase in a specific tissue, organ, or cell type (i.e., tissue-specific promoters) or otherwise under more precise environmental or developmental control or under the control of an inducible promoter. Examples of environmental conditions that can affect transcription include anaerobic conditions, elevated temperature, the presence of light, or chemical / hormone spray. For example, the disclosure incorporates the drought inducible promoter from maize (Busk (1997) supra); the cold, drought and high salinity inducible promoter of potato (Kirch (1997) Plant Mol. Biol. 33: 897 909).

In some embodiments, tissue-specific promoters promote transcription only within a certain time frame of the developmental stage within that tissue. See Blazquez (1998) Plant Cell 10: 791-800, with the characterization of the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J12: 367-77, which describes the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the AP1 gene for the identity of the floral meristem of A. thaliana; and Mandel (1995) Plant Molecular Biology, Volume 29, pp 995-1004, which describes the eIF4 meristem promoter. Tissue specific promoters that are active throughout the life cycle of a specific tissue can be used. In some embodiments, the nucleic acids disclosed herein are operably linked to an active promoter primarily only in cotton fiber cells. In some embodiments, the nucleic acids disclosed herein are operably linked to an active promoter primarily during the cotton fiber cell elongation steps, as described by Rinehart (1996) supra. Nucleic acids can be operably linked to the Fb12A gene promoter to be expressed preferably in cotton fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci. United States 89: 5769-5773; John, et al., US Patent Nos. 5,608,148 and 5,602,321, which describe specific cotton fiber promoters and methods for the construction of transgenic cotton plants. Root specific promoters can also be used to express the nucleic acids disclosed herein. Examples of root specific promoters include the promoter of the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123: 39-60). Other promoters that can be used to express the nucleic acids disclosed herein include, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seminal coat-specific, or some combination thereof; a leaf specific promoter (see Busk (1997) Plant J. 11: 1285 1295, which describes a corn leaf specific promoter); the ORF13 promoter from Agrobacterium rhizogenes (which exhibits high root activity, see Hansen (1997) supra), a specific promoter from corn pollen (see Guerrero (1990) Mol. Gen. Genet. 224: 161-168); an active tomato promoter can be used during fruit ripening, the

senescence and abscission of the leaves and, to a lesser extent, of the flowers (see Blume (1997) Plant J. 12: 731 746); a pistil-specific promoter of the potato SK2 gene (see Ficker (1997) Plant Mol. Biol. 35: 425 431); the pea Blec4 gene, which is active in the epidermal tissue of vegetative and floral root tips of transgenic alfalfa, making it a useful tool to direct the expression of external genes with respect to the epidermal layer of shoots or fibers in active growth; the ovule-specific BEL1 gene (see Reiser (1995) Cell 83: 735-742, GenBank No. U39944); and / or the promoter in Klee, US Patent No. 5,589,583, which describes a plant promoter region that is capable of conferring high levels of transcription in meristematic tissue and / or rapidly dividing cells.

In some embodiments, plant promoters that are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids disclosed herein. For example, the method may use the auxin response element E1 promoter fragment (AuxREs) in soybeans (Glycine max L.) (Liu (1997) Plant Physiol. 115: 397-407); the Arabidopsis GST6 promoter that responds to auxin (also responds to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the tobacco auxin inducible parC promoter (Sakai (1996) Plant Cell Physiol. 37: 906-913); a plant biotin response element (Streit (1997) Mol. Plant. Microbe Interact. 10: 933-937); and the stress hormone responsive promoter, abscisic acid (Sheen (1996) Science 274: 1900-1902).

The nucleic acids disclosed herein can also be operably linked to plant promoters that are inducible upon exposure to chemical reagents that can be applied to the plant, such as herbicides or antibiotics. For example, the corn In2-2 promoter, activated by herbicidal protectors of benzenesulfonamide (De Veylder (1997) Plant Cell Physiol. 38: 568-577) can be used; the application of different herbicidal protectors induces differentiated gene expression patterns, including expression in the roots, hydatodes and the apical meristem of the outbreak. Coding sequences may be under the control of, for example, a tetracycline inducible promoter, such as that described with transgenic tobacco plants containing the arginine decarboxylase gene from Avena sativa L. (oats) (Masgrau (1997) Plant J. 11: 465-473); or an element that responds to salicylic acid (Stange (1997) Plant J. 11: 1315-1324). With the use of promoters induced by chemical agents (such as hormones or pesticides), that is, promoters that respond to a chemical agent that can be applied to the transgenic plant in the field, the expression of a polypeptide disclosed herein can be induced at a specific stage of plant development. Accordingly, the disclosure also provides transgenic plants that contain an inducible gene encoding polypeptides disclosed herein, whose host range is limited to the target plant species, such as corn, rice, barley, soybean, tomato, wheat, potato, and other crops, inducible at any stage of crop development.

The skilled artisan will recognize that a tissue-specific plant promoter can activate the expression of operably linked sequences in tissues other than target tissues. Accordingly, in some embodiments, a tissue specific promoter is one that activates expression preferably in the target tissue or cell type but can also cause expression in other tissues.

The nucleic acids disclosed herein can also be operably linked to plant promoters that are inducible upon exposure to chemical reagents. These reagents include, for example, herbicides, synthetic auxins, or antibiotics that can be applied, such as by spraying, on transgenic plants. The inducible expression of nucleic acids producing the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein will allow the farmer to select plants with the optimal expression and / or activity of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase. In this way the development of the plant parts can be controlled. In this way disclosure provides the means to facilitate the harvesting of plants and plant parts. For example, in several embodiments, the corn In2-2 promoter, activated by herbicidal benzenesulfonamide protectors (De Veylder (1997) Plant Cell Physiol. 38: 568-577) is used; the application of different herbicidal protectors induces differentiated gene expression patterns, including expression in the roots, hydatodes and the apical meristem of the outbreak. The coding sequences according to the invention are also under the control of, for example, a tetracycline inducible promoter, such as that described with transgenic tobacco plants containing the arginine decarboxylase gene from Avena sativa L. (oats) ( Masgrau (1997) Plant J. 11: 465-473); or an element that responds to salicylic acid (Stange (1997) Plant J. 11: 1315-1324).

In some embodiments, proper expression of the polypeptide may require a polyadenylation region at the 3 'end of the coding region. The polyadenylation region may be derived from the natural gene, from a variety of different plant or animal genes, or from another origin or from genes in the Agrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehicles comprising nucleic acids disclosed herein, such as sequences encoding aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein. The expression vectors and cloning vehicles disclosed herein may comprise viral particles, baculoviruses, phage, plasmids, phagemids, cosmids, fossids, artificial bacterial chromosomes, viral DNA (such as varicella vaccine, adenovirus, smallpox virus.

avian, pseudorrabia and SV40 derivatives), P1-based artificial chromosomes, yeast plasmids, and any other vector specific for specific hosts of interest (such as bacillus, Aspergillus, and yeast). Vectors disclosed herein can include chromosomal, non-chromosomal, and synthetic DNA sequences. A large number of suitable vectors are known to those of skill in the art and are commercially available. Exemplary vectors include: bacterial: pQE ™ vectors (Qiagen, Valencia, CA), pBLUESCRIPT ™ plasmids, pNH vectors, lambda-ZAP vectors (Stratagene, La Jolla, CA); ptrc99a, pKK223-3, pDR540, pRIT2T (GE Healthcare, Piscataway, N.J.), pET vectors (Novagen, Madison, Wis.); Eukaryotes: pXT1, pSG5 (Stratagene, La Jolla, CA), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector can be used as long as it is replicable and viable in the host. Low copy or high copy vector vectors can be used in the present invention. The "plasmids" can be commercially available, publicly available without restrictions or can be constructed from available plasmids according to published procedures. Equivalent plasmids to those described herein are known in the art and these will be apparent to those skilled in the art.

The expression vector may comprise a promoter, a ribosome binding site to initiate translation, and a transcription terminator. The vector can also include appropriate sequences to amplify expression. Mammalian expression vectors can comprise an origin of replication, all necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5 'flanking non-transcribed sequences. In some embodiments, DNA sequences derived from the SV40 junction and polyadenylation sites can be used to provide the necessary non-transcribed genetic elements.

In some embodiments, expression vectors contain one or more selectable marker genes to allow selection of vector-containing host cells. Such selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance to eukaryotic cell cultures, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRP1 gene. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.

In some embodiments, vectors to express the polypeptide or fragment thereof in eukaryotic cells contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA that can be from about 10 to about 300 bp in length. They can act on a promoter to increase its transcription. Exemplary enhancers include the SV40 enhancer on the late side of the origin of replication bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus promoters.

A nucleic acid sequence can be inserted into a vector by a variety of procedures. In general, the sequence is ligated to the desired position in the vector after digestion of the insert and the vector with the appropriate restriction endonucleases. Alternatively, blunt ends can be attached to the insert and vector. A variety of cloning techniques are known in the art, such as those described in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2a Ed., Cold Spring Harbor Laboratory Press (1989). Such procedures and others are considered to be within the scope of those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, non-chromosomal, and synthetic DNA sequences derived from SV40; Bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as variola vaccine, adenovirus, fowlpox virus, and pseudorrabia. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook.

Specific bacterial vectors that can be used include the commercially available plasmids comprising genetic elements of the known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, Wis ., USA) pQE70, pQE60, pQE-9 (Qiagen, Valencia, CA), pD10, psiX174 pBLUESCRIPT II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene, La Jolla, CA), ptrc99a, pKK223 , pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8, pET (Novagen, Madison, Wis.) and pCM7. Specific prokaryotic vectors include pSV2CAT, pOG44, pXT1, pSG (Stratagene, La Jolla, CA) pSVK3, pBPV, pMSG and pSVL (Pharmacia). However, any other vector can be used as long as it is replicable and viable in the host cell.

The nucleic acids disclosed herein can be expressed in expression cassettes, vectors, or viruses and can be expressed transiently or stably in plant cells and seeds. An exemplary transient expression system uses episomal expression systems, such as cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcribing an episomal minichromosome containing supercoiled DNA, see Covey (1990) Proc. Natl. Acad. Sci. United States 87: 1633-1637. Alternatively, the coding sequences, i.e., all or sub-fragments of sequences disclosed herein, may

insert itself into the genome of a plant host cell becoming an integral part of the host chromosomal DNA. Sense or antisense transcripts can be expressed in this way. A vector comprising the sequences (such as promoters or coding regions) of nucleic acids disclosed herein can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed. For example, the marker may encode biocidal resistance, such as antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicidal resistance, such as resistance to chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins in plants are known in the art and may include, for example, Agrobacterium spp., Potato virus X vectors (see Angell (1997) EMBO J. 16: 3675-3684 ), tobacco mosaic virus (see Casper (1996) Gene 173: 69-73), tomato branched dwarf virus (see Hillman (1989) Virology 169: 42-50), tobacco print virus (see Dolja ( 1997) Virology 234: 243252), golden bean mosaic virus (see Morinaga (1993) Microbiol Immunol. 37: 471-476), cauliflower mosaic virus (see Cecchini (1997) Mol. Plant. Microbe Interact. 10 : 1094-1101), Ac / Ds transposable element of maize (see Rubin (1997) Mol. Cell. Biol. 17: 6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204: 161-194), and maize suppressor-mutant transposable element (Spm) (see Schlappi (1996) Plant Mol. Biol. 32: 717-725); and derivatives thereof.

In some embodiments, the expression vector can have two replication systems to allow it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Additionally, to integrate expression vectors, the expression vector can contain at least one sequence homologous to the genome of the host cell. It can contain two homologous sequences that flank the expression construct. The integration vector can be targeted to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are known in the art.

The expression vectors disclosed herein can also include a selectable marker gene to allow selection of bacterial strains that have been transformed, such as genes that provide bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, and tetracycline. . Selectable markers can also include biosynthetic genes, such as those from the histidine, tryptophan, and leucine biosynthetic pathways.

The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (s) (promoter) to direct RNA synthesis. The so-called bacterial specific promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include the early CMV, the thymidine kinase from HSV, the early and late SV40, the LTR from retroviruses, and the mouse metallothionein-I. Selection of the appropriate vector and promoter is within the level of skill in the art. The expression vector also contains a ribosome binding site to initiate translation and a transcription terminator. The vector can also include appropriate sequences to amplify expression. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers. Additionally, expression vectors In some embodiments contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture or such as tetracycline or ampicillin resistance in E .coli.

Mammalian expression vectors can also comprise an origin of replication, all necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5 'flanking non-transcribed sequences. In some embodiments, DNA sequences derived from the SV40 junction and polyadenylation sites can be used to provide the necessary non-transcribed genetic elements.

Vectors to express the polypeptide or fragment thereof in eukaryotic cells can also contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA, generally about 10 to about 300 bp in length, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the origin of replication bp 100-270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus promoters.

Furthermore, expression vectors can contain one or more selectable marker genes to allow selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance to eukaryotic cell cultures, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRP1 gene.

In some embodiments, the nucleic acid encoding one of the polypeptides disclosed herein and sequences substantially identical thereto or fragments comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more consecutive amino acids thereof are assembled in the appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof. In

In some embodiments, the nucleic acid can encode a fusion polypeptide in which one of the polypeptides according to the invention or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more consecutive amino acids thereof are fused to heterologous peptides or polypeptides, such as N-terminus identification peptides that impart desired characteristics, such as increased stability or simplified purification.

The appropriate DNA sequence can be inserted into the vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector after digestion of the insert and the vector with the appropriate restriction endonucleases. Alternatively, blunt ends can be attached to the insert and vector. A variety of cloning techniques are disclosed in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2a Ed., Cold Spring Harbor Laboratory Press (1989). Such procedures and others are considered to be within the scope of those skilled in the art.

The vector may, for example, be in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, non-chromosomal, and synthetic DNA sequences derived from SV40; Bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as variola vaccine, adenovirus, fowlpox virus, and pseudorrabia. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host cells and transformed cells

The invention also provides transformed cells comprising nucleic acid sequences disclosed herein, such as sequences encoding aldolase enzymes, such as pyruvate aldolases, HMG and / or KHG aldolase disclosed herein or vectors disclosed herein. The host cell can be any of the host cells known to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include any Streptomyces, Staphylococcus, Pseudomonas, or Bacillus species, including E. coli, Bacillus subtilis, Pseudomonas fluorescens, Bacillus cereus, or Salmonella typhimurium. Exemplary fungal cells include any Aspergillus species. Exemplary yeast cells include any species of Pichia, Saccharomyces, Schizosaccharomyces, or Schwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae

or Schizosaccharomyces pombe. Exemplary insect cells include any Spodoptera or Drosophila species, including DrosophilaS2 and Spodoptera Sf9. Exemplary animal cells include the CHO, COS, or Bowes melanoma cell lines or any murine or human cell lines. Selection of an appropriate host is within the capabilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are known and described in the technical and scientific literature. See Weising (1988) Ann. Rev. Genet. 22: 421-477; United States Patent No. 5,750,870.

The vector can be introduced into host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

In some embodiments, the nucleic acids or vectors disclosed herein are introduced into cells for detection, therefore, nucleic acids enter cells adequately for subsequent expression of the nucleic acid. The method of introduction largely depends on the target cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, lipofection (such as LIPOFECTIN ™), electroporation, viral infection, etc. Candidate nucleic acids can stably integrate the host cell genome (for example, with retroviral introduction) or can exist transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, using standard regulatory sequences, selection markers , etc.). Since many pharmaceutically important detections require human or model mammalian target cells, retroviral vectors capable of transfecting such targets can be used.

When appropriate, the engineered host cells can be grown in conventional nutrient media modified as appropriate to activate promoters, select transformants, or amplify the genes disclosed herein. After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter can be induced by appropriate means (such as temperature change or chemical induction) and the cells can be cultured for an additional period to allowing them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, homogenized by physical or chemical means, and the resulting crude extract is preserved for further purification. Microbial cells used for protein expression can be homogenized by any convenient method, including cycling

freezing and thawing, exposure to ultrasound, mechanical homogenization or use of cell lysis agents. Such methods are known to those skilled in the art. The expressed polypeptide or fragment thereof can be collected and purified from the recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography. , hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, to complete the configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be used for the final stages of purification.

The constructs in host cells can be used in a conventional way to produce the gene product encoded by the recombinant sequence. Depending on the host employed in a recombinant production procedure, polypeptides produced by host cells containing the vector can be glycosylated

or they can be non-glycosylated. The polypeptides disclosed herein may or may not also include an initial methionine amino acid residue.

In vitro translation systems can also be employed to produce a polypeptide disclosed herein. In vitro translation systems can use mRNA transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some embodiments, the DNA construct can be linearized before carrying out an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate in vitro translation extract, such as an extract from rabbit reticulocytes, to produce the desired polypeptide or fragment thereof.

Expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture or such as tetracycline or ampicillin resistance in E. coli.

Host cells containing the polynucleotides of interest, such as nucleic acids disclosed herein, can be cultured in nutrient media as modified to appropriate promoters, select transformants, or amplify genes. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to one skilled in the art. Clones identified as having the enzyme activity specified below can be sequenced to identify the polynucleotide sequence encoding an enzyme that has enhanced activity.

The disclosure provides methods for overexpressing aldolase enzymes, such as pyruvate aldolases, such as recombinant HMG and / or KHG aldolase in cells comprising and expressing a vector comprising a nucleic acid disclosed herein, such as a nucleic acid comprising a sequence of nucleic acids with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or greater sequential identity with a sequence disclosed herein in a region of at least about 100 residues, where the sequential identities are determined by analysis with a sequential comparison algorithm

or by visual inspection, or a nucleic acid that hybridizes under stringent conditions to a nucleic acid sequence disclosed herein or a subsequence thereof. Overexpression can occur by any means, such as the use of a highly active promoter, a dicistronic vector, or by gene amplification of the vector.

The nucleic acids disclosed herein can be expressed or overexpressed in any in vitro or in vivo expression system. Any cell culture system can be used to express or overexpress recombinant proteins, including bacterial, insect, yeast, fungal, or mammalian cultures. Overexpression can occur through the appropriate choice of promoters, enhancers, vectors (such as the use of replicon vectors, dicistronic vectors (see Gurtu (1996) Biochem. Biophys. Res. Commun. 229: 2958)), media, systems of cultivation and the like. In some embodiments, gene amplification using selection markers, such as glutamine synthetase (see Sanders (1987) Dev. Biol. Stand. 66: 55-63), is used in cellular systems to overexpress the polypeptides disclosed herein.

Additional details regarding this approach are found in public literature and / or are known to experts. In a specific non-exhaustive example, said publicly available literature includes EP 0659215 (WO 9403612 A1) (Nevalainen et al.); Lapidot, A., Mechaly, A., Shoham, Y., "Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6", J. Biotechnol. November 51: 259-64 (1996); Lüthi, E., Jasmat, N. B., Bergquist, P. L., “Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum: overexpression of the gene in Escherichia coli and characterization of the gene product”, Appl. Environ. Microbiol. September 56: 2677-83 (1990); and Sung, W. L., Luk, C. K., Zahab, D. M., Wakarchuk, W., "Overexpression of the Bacillus subtilis and circulans xylanases in Escherichia coli", Protein Expr. Purif. June 4: 2006 (1993), although these references do not describe the inventive enzymes of the present application.

The host cell can be any of the host cells known to those skilled in the art, including prokaryotic cells, eukaryotic cells, mammalian cells, insect cells, or plant cells. As representative examples of suitable hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces and Staphylococcus, fungal cells, such as Aspergillus, yeast such as any species of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe, cells of insects such as Drosophila S2 and Spodoptera Sf9, cells of animals such as CHO, COS, or Bowes melanoma and adenovirus. Selection of an appropriate host is within the capabilities of those skilled in the art.

The vector can be introduced into host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

When appropriate, the manipulated host cells can be grown in standard nutrient media modified as appropriate to activate promoters, select transformants, or amplify genes according to the invention. After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter can be induced by appropriate means (such as temperature change or chemical induction) and the cells can be cultured for an additional period to allowing them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, homogenized by physical or chemical means, and the resulting crude extract is preserved for further purification. Microbial cells used for protein expression can be homogenized by any convenient method, including freeze-thaw cycling, ultrasound exposure, mechanical homogenization, or use of cell lysing agents. Such methods are known to those skilled in the art. The expressed polypeptide or fragment thereof can be collected and purified from the recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, chromatography. affinity, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, to complete the configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be used for the final stages of purification.

Various mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include monkey kidney fibroblast COS-7 lines (described by Gluzman, Cell, 23: 175, 1981) and other cell lines capable of expressing proteins from a compatible vector, such as C127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional way to produce the gene product encoded by the recombinant sequence. Depending on the host employed in a recombinant production procedure, polypeptides produced by host cells containing the vector can be glycosylated

or they can be non-glycosylated. Polypeptides according to the invention may or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides disclosed herein or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more consecutive amino acids thereof can be synthetically produced by peptide synthesizers conventional, such as those described below. In other embodiments, fragments or portions of the polypeptides can be used to produce the corresponding full-length polypeptides by peptide synthesis; therefore, the fragments can be used as intermediates to produce the full length polypeptides.

In vitro translation systems can also be used to produce one of the polypeptides disclosed herein or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 or more. Consecutive amino acids thereof using mRNA transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some embodiments, the DNA construct can be linearized before carrying out an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate in vitro translation extract, such as an extract from rabbit reticulocytes, to produce the desired polypeptide or fragment thereof.

Nucleic acid amplification

By practicing the disclosure, nucleic acids disclosed herein and nucleic acids encoding aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, or modified nucleic acids disclosed herein, can be produced. by amplification, such as PCR. Amplification can also be used to clone or modify the nucleic acids disclosed in the

Present. Accordingly, the invention provides pairs of amplification primer sequences for amplifying nucleic acids disclosed herein. One skilled in the art can design pairs of amplification primer sequences for any part or for the full length of these sequences.

In some embodiments, the invention provides nucleic acids amplified by amplification primer pairs disclosed herein, such as primer pairs as set forth in approximately the first (5 ') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleic acid residues disclosed herein and in approximately the first (5 ') 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more residues of the complementary chains. In some embodiments, the disclosure provides amplification primer sequence pairs for amplifying a nucleic acid encoding a polypeptide having an enzyme aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, wherein the primer pair it is capable of amplifying a nucleic acid comprising a sequence disclosed herein or fragments or subsequences thereof. One or each member of the pair of amplification primer sequences may comprise an oligonucleotide comprising at least about 10 to 50 or more consecutive bases in the sequence or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more consecutive bases in the sequence. In some embodiments, the invention provides amplification primer pairs, wherein the primer pair comprises a first member having a sequence as set forth in approximately the first (5 ') 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of a nucleic acid disclosed herein and a second member having a sequence as established in approximately the first (5 ') 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more residues of the complementary chain of the first member.

The invention provides aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase generated by amplification, such as polymerase chain reaction (PCR), using a pair of amplification primers disclosed herein. In some embodiments, the invention provides methods for making an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase by amplification, such as PCR, using a pair of amplification primers disclosed herein. In some embodiments, the pair of amplification primers amplifies a nucleic acid from a library, such as a library, such as an environmental library.

Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (such as applying it to an array or a filter membrane), detect the nucleic acid or quantify the amount of a specific nucleic acid in a sample. In some embodiments, the isolated message is amplified from a cell or a cDNA library.

The person skilled in the art can select and design suitable oligonucleotide amplification primers. Amplification methods are also known in the art and include polymerase chain reaction, PCR (see PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, NY (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., NY, Ligase Chain Reaction (CSF) (see Wu (1989) Genomics 4: 560; Landegren (1988) Science 241: 1077; Barringer (1990) Gene 89: 117) ; transcription amplification (see Kwoh (1989) Proc. Natl. Acad. Sci. United States 86: 1173); and self-sustained sequence replication (see Guatelli (1990) Proc. Natl. Acad. Sci. United States 87: 1874) ; Q beta replicase amplification (see Smith (1997) J. Clin. Microbiol. 35: 1477-1491), automated Q beta replicase amplification assay (see Burg (1996) Mol. Cell. Probes 10: 257-271) and other RNA polymerase mediated techniques (such as NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152: 307-316; Ausubel et al. Current Protocols in Molecu Lar Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989). United States Patent Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13: 563-564.

Determination of sequential identity in nucleic acids and polypeptides

The disclosure provides nucleic acids that comprise sequences that are at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or more or total (100%) sequential identity (homology) with a nucleic acid disclosed herein (see Sequence Listing) in a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more waste. In some embodiments, the disclosure provides polypeptides comprising sequences that are at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more or total (100%) sequential identity with a polypeptide disclosed herein (see Sequence Listing). The degree of sequential identity (homology) can be determined using any computer program and associated parameters, including those described herein, such as BLAST

2.2.2. o FASTA version 3.0t78, with default parameters.

The nucleic acid sequences disclosed herein can comprise at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 or more consecutive nucleotides of a disclosed sequence herein and sequences basically identical thereto. The homologous sequences and nucleic acid sequence fragments disclosed herein can refer to sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequential identity (homology) with these sequences. Homology (sequential identity) can be determined using any of the software and parameters described herein, including FASTA version 3.0t78 with the default parameters. Homologous sequences also include RNA sequences in which uridines replace thymine in the nucleic acid sequences disclosed herein. Homologous sequences can be obtained using any of the procedures described herein or can result from correction of a sequencing error. It will be appreciated that the nucleic acid sequences disclosed herein can be represented in the traditional single-character format (See inside back cover of Stryer, Lubert. Biochemistry, 3rd Ed.,

W. H Freeman & Co., New York.) Or in any other format that records the identity of nucleotides in a sequence.

In some embodiments, the sequence comparison programs identified herein are used in this regard, that is, to determine if a nucleic acid or polypeptide sequence is within the scope of the disclosure. However, the sequential identities of proteins and / or nucleic acids (homologies) can be evaluated using any algorithm or sequential comparison program known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (see Pearson and Lipman, Proc. Natl. Acad. Sci. United States 85 (8): 2444-2448, 1988; Altschul et al.,

J. Mol. Biol. 215 (3): 403-410, 1990; Thompson Nucleic Acids Res. 22 (2): 4673-4680, 1994; Higgins et al., Methods Enzymol. 266: 383-402, 1996; Altschul et al., J. Mol. Biol. 215 (3): 403-410, 1990; Altschul et al., Nature Genetics 3: 266-272, 1993).

In some embodiments, homology or identity is measured using a sequence analysis software (such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Said computer program matches similar sequences assigning degrees of homology to various deletions, substitutions and other modifications. In some embodiments, the terms "homology" and "identity" in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid or nucleotide residues that are the same when comparing and aligning to determine the maximum match in a comparison window or designated region as measured using any number of sequential comparison algorithms or by manual alignment and visual inspection. In some embodiments, for sequential comparison, a sequence acts as a reference sequence against which the test sequences are compared. When using a sequential comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the parameters of the sequence algorithm program are designated. Default program parameters can be used or alternative parameters can be designated. The sequential comparison algorithm below calculates the percentage sequential identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window" as used herein includes the reference to a segment of any of the number of contiguous positions selected from the group consisting of from 20 to 600, generally about 50 to about 200, more generally about 100 to about 150 in which one sequence can be compared to the reference sequence with the same number of contiguous positions after optimally aligning the two sequences. Sequence alignment methods for comparison are known in the art. Optimal sequence alignment for comparison can be carried out, for example, by the Smith & Waterman local homology algorithm, Adv. Appl. Math. 2: 482, 1981, using the Needleman & Wunsch, J. Mol. Homology alignment algorithm. Biol. 48: 443, 1970, by the similarity search method of person & Lipman, Proc. Nat'l. Acad. Sci. United States 85: 2444, 1988, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) Or by manual alignment and visual inspection. Other algorithms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Multiple Aligned Sequence Analysis), AMPS (Alignment of Multiple Protein Sequences), ASSET (Aligned Segment Statistical Assessment Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (Block Enhanced Finder), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman's algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequential Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Comparison

Sensitive Sequential), LALIGN (Local Sequential Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction and Analysis Workstation), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Induced Multi-Sequence Alignment by Pattern), SAGA (Sequential Alignment Using Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences that have substantially identical sequences. There are several genome databases available, for example a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995). At least twenty-one different genomes have already been sequenced, including for example from M. genitalium (Fraser et al., Science 270: 397-403 (1995)), M. jannaschii (Bult et al., Science 23: 1058-73 ( 1996)), H. influenzae (Fleischmann et al., Science 269: 496-512 (1995)), E. coli (Blattner et al., Science 277: 1453-74 (1997)) and yeast (S. cerevisiae) (Mewes et al., Nature 387: 7-65 (1997)) and D. melanogaster (Adams et al., Science287: 2185-95 (2000)). Considerable progress has also been made in the sequencing of the genomes of model organisms, such as the mouse, C. elegansand Arabadopsis sp. Different organizations maintain several databases that contain annotated genomic information with some functional information and can be accessed through the Internet.

In some embodiments, the BLAST and BLAST 2.0 algorithms are used, which are described in Altschul et al., Nuc. Acids Res. 25: 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215: 403-410, 1990, respectively. The software for carrying out the BLAST analyzes is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match or reach some T-score threshold with a positive value when aligned with a word of the same length. in a sequence in the database. T refers to the near word score threshold (Altschul et al., Supra). These initial close-word hits act as seeds to start searches to find longer HSPs that contain them. Word hits extend both ways throughout each sequence to the extent that the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the M parameters (premium score for a pair of matched residues; always> 0). For amino acid sequences, a score matrix is used to calculate the cumulative score. The extension of the word hits in each direction can be stopped when: the accumulated alignment score falls to the quantity X from its maximum value reached; the cumulative score drops to zero or below, due to the accumulation of one or more residual negative score alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default values a word length (W) of 11, an expectation (E) of 10, M = 5, N = −4 and a comparison of both strings. For amino acid sequences, the BLASTP program uses as default values a word length of 3 and expectations (E) of 10 and the score matrix BLOSUM62 (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. United States 89: 10915 , 1989) alignments (B) of 50, expectation (E) of 10, M = 5, N = −4 and a comparison of both chains.

The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873, 1993). A measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability of a random match occurring between two nucleotides or amino acids. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2. In some embodiments less than at least about 0.01 and in other embodiments less than about 0.001.

In some embodiments, sequential protein and nucleic acid homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST"). Specifically, five specific BLAST programs are used to accomplish the following task:

(one)

 BLASTP and BLAST3 compare the query amino acid sequence with a database of protein sequences;

(2)

 BLASTN compares a query nucleotide sequence to a nucleotide sequence database;

(3)

 BLASTX compares the conceptual translation products of six frames of a query nucleotide sequence (both strands) with a protein sequence database;

(4)

 TBLASTN compares a query protein sequence with a translated nucleotide sequence database in all six reading frames (both strands); and

(5)

 TBLASTX compares translations in all six frames of a query nucleotide sequence to translations in all six frames of a nucleotide sequence database.

BLAST programs identify homologous sequences by identifying similar segments, referred to herein as "high-scoring segment pairs", between a query amino acid or nucleic acid sequence and a test sequence which, in some embodiments, is derived from a nucleic acid or protein sequence database. The high scoring segment pairs, in some embodiments, are identified (ie, aligned) by a scoring matrix, many of which are known in the art. In some embodiments, the scoring matrix used is the BLOSUM62 matrix (Gonnet (1992) Science 256: 1443-1445; Henikoff and Henikoff (1993) Proteins 17: 49-61). In some embodiments, PAM or PAM250 matrices may also be used (see Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation). BLAST programs can be accessed through the U.S. National Library of Medicine.

The parameters used with the aforementioned algorithms can be adapted depending on the sequence length and degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of user instructions.

Computer systems and software products

The disclosure describes computers, computer systems, computer readable media, computer program products, and the like in which the nucleic acid and polypeptide sequences disclosed herein are recorded or stored. Additionally, by practicing the methods disclosed herein, such as to determine and identify sequential identities (to determine if a nucleic acid is within the scope of the invention), structural homologies, motifs, and the like in silico, one can store, record and handle a nucleic acid or polypeptide sequence disclosed herein in any medium that can be read or accessed by a computer.

As used herein, the terms "recorded" and "stored" refer to the process of storing information on a computer medium. An expert can easily adopt any known method of recording information on a computer-readable medium to generate products comprising one or more nucleic acid and / or polypeptide sequences disclosed herein. As used herein, the terms and terms "computer", "computer program" and "processor" are used in their broader general contexts and incorporate all of the devices described in detail below. A "coding sequence for" or a "sequence encoding" a specific polypeptide or protein, is a nucleic acid sequence that can be transcribed and translated to a polypeptide or protein when placed under the control of appropriate regulatory sequences.

The polypeptides disclosed herein include sequences disclosed herein and sequences substantially identical thereto and enzymatically active subsequences and fragments of any of the preceding sequences. In some embodiments, substantially identical or homologous polypeptide sequences refer to polypeptide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or total (100%) sequential identity (homology) with the sequence disclosed herein.

Homology (sequential identity) can be determined using any of the computer programs and parameters described herein. A nucleic acid or polypeptide sequence disclosed herein can be stored, recorded, and handled in any medium that can be read or accessed by a computer. As used herein, the terms "recorded" and "stored" refer to the process of storing information on a computer medium. A skilled person can easily adopt any of the currently known methods for recording information on a computer readable medium to generate products comprising one or more of the nucleic acid sequences disclosed herein, one or more of the polypeptide sequences disclosed herein. Present. Another embodiment is a computer readable medium having at least 2, 5, 10, 15 or 20 or more nucleic acid or polypeptide sequences disclosed herein recorded.

In addition, a computer readable medium is described having one or more nucleic acid sequences disclosed herein recorded. Also disclosed is a computer readable medium that has one or more polypeptide sequences disclosed herein recorded on it. Also disclosed is a computer readable medium having at least 2, 5, 10, 15 or 20 or more of the nucleic acid or polypeptide sequences recorded as set forth above.

Computer readable media includes magnetically readable media, optically readable media, electronically readable media, and magnetic / optical media. For example, computer-readable media can be a hard drive, floppy disk, magnetic tape, CD-ROM, digital versatile disc (DVD), random access memory (RAM), or read-only memory (ROM) as well as also other types of other means known to those skilled in the art.

Systems (such as Internet-based systems), such as computer systems that store and manage the sequential information described herein, are also disclosed. An example of a computer system

100 is illustrated in block diagram format in FIG. 9. As used herein, "a computer system" refers to the equipment components, programming components, and data storage components used to analyze a nucleotide sequence of a nucleic acid sequence disclosed in the present or a polypeptide sequence disclosed herein. In some embodiments, computer system 100 includes a processor for processing, accessing, and handling sequence data. Processor 105 any known type of central processing unit, such as, for example, the Pentium III from Intel Corporation or similar processors from Sun, Motorola, Compaq, AMD or International Business Machines.

In some embodiments, computer system 100 is a general-purpose system comprising processor 105 and one or more internal data storage components 110 for storing data and one or more data recovery devices for retrieving data stored in the components. data storage. An expert can easily appreciate that any of the currently available computer systems is adequate.

In one embodiment, the computer system 100 includes a processor 105 connected to a bus that is connected to a main memory 115 (in an embodiment implemented as RAM) and one or more internal data storage devices 110, such as a hard disk and / or another computer-readable medium that has data recorded on it. In some embodiments, computer system 100 further includes one or more data recovery devices 118 for reading data stored on internal data storage devices 110.

Data recovery device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connecting to a remote data storage system (such as through Internet) etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, compact disc, magnetic tape, etc. that contains control logic and / or data recorded in it. The computer system 100 may advantageously include or be programmed by a suitable computer program to read the control logic and / or the data from the data storage component once it is inserted into the data recovery device.

The computer system 100 includes a screen 120 that is used to display the output data for the user of the computer. It should be further mentioned that the computer system 100 may be connected to other computer systems 125 a-c in a wide area network or network to provide centralized access to the computer system

100.

The computer program for accessing and processing the nucleotide sequences of a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein and sequences substantially identical thereto (such as search tools , comparison tools, and modeling tools, etc.) may reside in main memory 115 during execution.

In some embodiments, computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein and sequences substantially identical thereto. It is stored in a computer readable medium with a reference nucleotide or polypeptide sequence (s) stored in a computer readable medium. A "sequence comparison algorithm" refers to one or more programs that are implemented (locally or remotely) in a computer system 100 to compare a nucleotide sequence with other nucleotide and / or compound sequences stored within a storage medium of data. For example, the sequence comparison algorithm can compare the nucleotide sequences of a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein and sequences substantially identical thereto, stored. on a computer readable medium with reference sequences stored on a computer readable medium to identify homologies or structural motifs.

Figure 10 is a flowchart illustrating an embodiment of a process 200 for comparing a new nucleotide or protein sequence to a sequence database to determine levels of homology between the new sequence and the sequences in the database. . The sequence database may be a private database stored within a computer system 100 or a public database such as GENBANK that is available through the Internet.

Process 200 starts in an initial state 201 and then goes into a state 202 where the new sequence to be compared is stored in memory in a computer system 100. As discussed above, the memory could be any type of memory , including a RAM or an internal storage device.

The process 200 then enters a state 204 where a sequence database is opened to carry out the analysis and comparison. Then process 200 goes to a state 206 where the first sequence stored in the database is read from memory in the computer. A comparison is then carried out in a state 210 to determine if the first sequence equals the second sequence. It should be noted that this stage is not limited to carrying out an exact comparison between the new sequence and the first sequence in the database. Methods of comparing two nucleotide or protein sequences are known to those skilled in the art, even if they are not identical. For example, gaps can be introduced into a sequence to raise the level of homology between two evaluated sequences. Parameters that control whether gaps or other characteristics are introduced to a sequence during comparison are generally entered by the computer system user.

After the comparison of the two sequences in state 210 has been carried out, in decision state 210 it is determined whether the two sequences are the same. Of course, the term "equal" is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as "equal" in process 200.

If the two sequences are determined to be the same, process 200 goes to a state 214 where the sequence name of the database is displayed to the user. This status notifies the user that the sequence with the displayed name complies with the homology restrictions that were entered. Once the name of the stored sequence is displayed to the user, the process 200 goes to a decision state 218 where it is determined if there are more sequences in the database. If there are no more sequences in the database, then process 200 ends in a final state 220. However, if there are more sequences in the database, then process 200 goes to a state 224 where it shifts. a pointer to the next sequence in the database so that it can be compared to the new sequence. In this way, the new sequence is aligned and compared to each sequence in the database.

It should be noted that if the sequences were determined to be non-homologous in decision state 212, then process 200 will immediately go to decision state 218 to determine if other sequences are available in the database for comparison.

Accordingly, there is disclosed a computer system comprising a processor, a data storage device having stored therein a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein. and sequences substantially identical thereto, a data storage device having recoverable nucleotide sequences or polypeptide sequences stored therein for comparison with a nucleic acid sequence disclosed herein and sequences substantially identical thereto. the same or a polypeptide sequence disclosed herein and sequences substantially identical thereto and a sequence comparator for carrying out the comparison. The sequence comparator can indicate a level of homology between the compared sequences or identify structural motifs in the nucleic acid code described above, a nucleic acid sequence disclosed herein and sequences substantially identical thereto, or a polypeptide sequence disclosed in the present and sequences substantially identical thereto or can identify structural motifs in sequences that are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored sequences of at least 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acid sequences disclosed herein and sequences substantially identical to the same or the polypeptide sequences disclosed herein and sequences substantially identical thereto.

There is also disclosed a method of determining the level of homology between a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein and sequences substantially identical thereto and a nucleotide sequence of reference. The method includes reading the nucleic acid code or polypeptide code and the reference nucleotide or polypeptide sequence through the use of a computer program that determines levels of homology and determining homology between the nucleic acid code or code. of polypeptides and the reference nucleotide or polypeptide sequence with the computer program. The computer program may be any of several computer programs for determining homology levels, including those specifically indicated herein (such as BLAST2N with predetermined parameters or with any modified parameters). The method can be implemented using the computer systems described above. The method can also be carried out by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the nucleic acid sequences disclosed herein and sequences substantially identical thereto or the disclosed polypeptide sequences herein and sequences substantially identical thereto through the use of the computer program and determining homology between the nucleic acid codes or polypeptide codes and the reference nucleotide sequences or polypeptide sequences.

FIG. 11 is a flowchart illustrating an embodiment of a process 250 on a computer for determining whether two sequences are homologous. Process 250 starts in an initial state 252 and then goes to a

state 254 where a first sequence to be compared is stored in memory. The second sequence to be compared is then stored in memory in a 256 state. Then process 250 goes to a state 260 where the first character is read in the first sequence and then goes to a state 262 where it is read the first character of the second sequence. It will be understood that if the sequence is a nucleotide sequence then the character will normally be A, T, C, G or U. If the sequence is a protein sequence then it is in the single letter amino acid code so that the first and second sequences can be easily compared.

Then it is determined in a decision state 264 whether the two characters are the same. If they are the same, then process 250 enters a state 268 where the following characters are read in the first and second sequences. Then it is determined whether the following characters are the same. If they are, then process 250 continues this cycle until two characters are not the same. If it is determined that the next two characters are not the same, process 250 goes into decision state 274 to determine if there are more characters to read in the sequences.

If there are no more characters to read, then process 250 goes to a state 276 where the level of homology between the first and second sequences is displayed to the user. The homology level is determined by calculating the proportion of equal characters between the sequences with respect to the total number of sequences in the first sequence. Therefore, if each character in a first sequence of 100 nucleotides were aligned with each character in a second sequence, the level of homology would be 100%.

Alternatively, the computer program may be a computer program that compares the nucleotide sequences of a nucleic acid sequence as set forth herein with one or more reference nucleotide sequences to determine whether the nucleic acid code disclosed herein. and the sequences substantially identical thereto differ from a reference nucleic acid sequence in one or more positions. Optionally said program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of the reference polynucleotides or a nucleic acid sequence disclosed herein and sequences substantially identical thereto. In some embodiments, the computer program may be a program that determines whether a nucleic acid sequence disclosed herein and sequences substantially identical thereto contain a single nucleotide polymorphism (SNP) with respect to a reference nucleotide sequence .

Therefore, another embodiment of the invention is a method of determining whether a nucleic acid sequence disclosed herein and sequences substantially identical thereto differ by one or more nucleotides from a reference nucleotide sequence comprising the steps of read the nucleic acid code and the reference nucleotide sequence through the use of a computer program that identifies differences between nucleic acid sequences and identify differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program that identifies polymorphisms in a single nucleotide. The method can be implemented by the computer systems described above and the method illustrated in Figure 11. The method can also be carried out by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the sequences of Nucleic acids disclosed herein and sequences substantially identical thereto and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program.

In other embodiments, the computer-based system may further comprise an identifier for identifying features in a nucleic acid sequence disclosed herein or a polypeptide sequence disclosed herein and sequences substantially identical thereto. An "identifier" refers to one or more programs that identify certain characteristics within a nucleic acid sequence disclosed herein or a polypeptide sequence disclosed herein. In some embodiments, the identifier may comprise a program that identifies an open reading frame in a nucleic acid sequence disclosed herein and sequences substantially identical thereto.

FIG. 12 is a flow chart illustrating an embodiment of an identifier process 300 for detecting the presence of a feature in a sequence. Process 300 starts in an initial state 302 and then goes into state 304 where a first sequence to be verified to determine its characteristics is stored in memory 115 in computer system 100. Process 300 then goes into a state 306 where a sequence characteristics database is opened. This database will include a list of each attribute of the characteristics together with the name of the characteristic. For example, a feature name could be "Initiation Codon" and the attribute would be "ATG". Another example would be the feature name "TAATAA sequence" and the feature attribute would be "TAATAA". An example of such a database is that produced by the University of Wisconsin Genetics Computer Group. Alternatively, the features may be structural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzyme active sites, helix-gyro-helix motifs, or other motifs known to those skilled in the art.

Once the feature database is opened in state 306, process 300 goes to state 308 where the first feature is read from the database. Next, a comparison is made of the attribute of the first characteristic with the first sequence in state 310. Then it is determined in a decision state 316 if the attribute of the characteristic was found in the first sequence. If the attribute was found, then process 300 goes to a state 318 where the name of the found feature is displayed to the user.

The process 300 then enters a decision state 320 where it is determined whether there are more features in the database. If there are no more characteristics, then process 300 ends in a final state 324. However, if there are more characteristics in the database, then process 300 reads the next sequence characteristic in a state 326 and returns to state 310. where the attribute of the following characteristic is compared with the first sequence. It should be noted that if the feature attribute is not found in the first sequence in decision state 316, process 300 goes directly to decision state 320 to determine if there are more features in the database.

Accordingly, there is also disclosed a method of identifying a characteristic within a nucleic acid sequence disclosed herein and sequences substantially identical thereto or a polypeptide sequence disclosed herein and sequences substantially identical thereto, comprising reading the nucleic acid code (s) or polypeptide code (s) through the use of a computer program that identifies features in it (s) and identify features within the nucleic acid code (s) with the computer program. In some embodiments, the computer program comprises a program that identifies open reading frames. The method can be carried out by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the nucleic acid sequences disclosed herein and sequences substantially identical to the same or the sequences of polypeptides disclosed herein and sequences substantially identical thereto, through the use of the computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program.

A nucleic acid sequence disclosed herein and sequences substantially identical thereto, or a polypeptide sequence disclosed herein and sequences substantially identical thereto, can be stored and managed in various data processing programs in a variety of formats. For example, a nucleic acid sequence disclosed herein and sequences substantially identical thereto, or a polypeptide sequence disclosed herein and sequences substantially identical thereto, may be stored as text in a word processing file, such as Microsoft WORD ™

or WORDPERFECT ™ or as an ASCII file in a variety of database programs known to those of skill in the art, such as DB2 ™, SYBASE ™ or ORACLE ™. In addition, many computer programs and databases can be used as sequence comparison algorithms, nucleotide sequence identifiers or sources, or reference polypeptide sequences to compare with a nucleic acid sequence disclosed herein and sequences substantially identical to that. itself or a polypeptide sequence disclosed herein and sequences substantially identical thereto. The following list is not intended to limit disclosure, but to provide guidance on programs and databases that are useful with the nucleic acid sequences disclosed herein and sequences substantially identical thereto.

or the polypeptide sequences disclosed herein and sequences substantially identical thereto.

Programs and databases that may be used include, but are not limited to: MACPATTERN ™ (EMBL), DISCOVERYBASE ™ (Molecular Applications Group), GENEMINE ™ (Molecular Applications Group), LOOK ™ (Molecular Applications Group), MACLOOK ™ (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al., J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. United States, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6: 237-245, 1990), CATALYST ™ (Molecular Simulations Inc.), Catalyst / SHAPE ™ (Molecular Simulations Inc.), Cerius2. DBAccess ™ (Molecular Simulations Inc.), HYPOGEN ™ (Molecular Simulations Inc.), INSIGHT II ™, (Molecular Simulations Inc.), DISCOVER ™ (Molecular Simulations Inc.), CHARMm ™ (Molecular Simulations Inc.), FELIX ™ ( Molecular Simulations Inc.), DELPHI ™, (Molecular Simulations Inc.), QuanteMM ™, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), MODELER ™ (Molecular Simulations Inc.), ISIS ™ (Mo lecular Simulations Inc.), Quanta / Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc. ), the MDL Available Chemicals Directory database, the MDL Drug Data Report database, the Comprehensive Medicinal Chemistry database, the Derwents World Drug Index database, the BioByteMasterFile database, the Genbank database and the Genseqn database. Many other programs and databases will be apparent to one skilled in the art, given the present disclosure.

Motifs that can be detected using the above-mentioned programs include sequences encoding leucine closures, helix-gyro-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides that direct secretion of the encoded proteins, sequences involved in the regulation of transcription such as homeosequences, acidic extensions, enzymatic active sites, substrate binding sites and enzymatic cleavage sites.

Nucleic Acid Hybridization

The disclosure provides isolated, synthetic, or recombinant nucleic acids that hybridize under stringent conditions to a sequence disclosed herein, such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 110, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ I D NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO : 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283 , SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO : 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333 , SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 or SEQ ID NO: 338. The stringent conditions may be extreme stringency conditions, medium stringency conditions and / or low stringency conditions, including the low and high stringency conditions described herein. In some embodiments, it is the stringency of the wash conditions that establishes the conditions that determine whether a nucleic acid is disclosed herein, as discussed below.

"Hybridization" refers to the process by which a nucleic acid chain binds to a complementary chain through base pairing. Hybridization reactions can be sensitive and selective so that a specific sequence of interest can be identified even in samples where it is at a low concentration. Suitably stringent conditions can be defined, for example, by the concentrations of salt or formamide in the prehybridization and hybridization solutions or by the hybridization temperature and are known in the art. In other embodiments, stringency can be increased by reducing the salt concentration, increasing the concentration of formamide, or by raising the hybridization temperature. In other embodiments, the nucleic acids disclosed herein are defined by their ability to hybridize under various stringency conditions (such as high, medium, and low), as set forth herein.

In some embodiments, hybridization under high stringency conditions comprises about 50% formamide at about 37 ° C to 42 ° C. In some embodiments, the hybridization conditions comprise low stringency conditions in about 35% to 25% formamide at about 30 ° C to 35 ° C. In some embodiments, hybridization conditions comprise high stringency conditions, such as at 42 ° C in 50% formamide, 5 × SSPE, 0.3% SDS, and 200 µg / ml denatured, sheared salmon sperm DNA . In some embodiments, hybridization conditions comprise these conditions of reduced stringency, but in 35% formamide at a reduced temperature of 35 ° C. The temperature range corresponding to a specific stringency level can be further narrowed by calculating the ratio of purine to pyrimidine of the nucleic acid of interest and adjusting the temperature accordingly. Variations in the ranges and conditions mentioned above are known in the art.

In other embodiments, the nucleic acids disclosed herein, as defined by their ability to hybridize under stringent conditions, may have between about five residues and the full length of the nucleic acid disclosed herein, such that they may have at least minus 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 , 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues in length. Nucleic acids shorter than full length are also included. These nucleic acids may be useful, for example, as hybridization probes, labeling probes, oligonucleotide probes for PCR, siRNA or miRNA (single-stranded or double-stranded), antisense, or sequences encoding antibody binding peptides ( epitopes), motifs, active sites and the like.

In some embodiments, the nucleic acids disclosed herein are defined by their ability to hybridize under stringent conditions comprising conditions of about 50% formamide at about 37 ° C to 42 ° C. In some embodiments, the nucleic acids disclosed herein are defined by their ability to hybridize under stringency conditions comprising conditions in about 35% to 25% formamide at about 30 ° C to 35 ° C.

Alternatively, the nucleic acids disclosed herein are defined by their ability to hybridize in high stringency comprising conditions at 42 ° C in 50% formamide, 5 × SSPE, 0.3% SDS and a repetitive sequence blocking nucleic acid , such as cot-1 or salmon sperm DNA (such as 200 µg / ml denatured, sheared salmon sperm DNA). In some embodiments, the nucleic acids disclosed herein are defined by their ability to hybridize under conditions of reduced stringency comprising 35% to 40% formamide at a reduced temperature of 35 ° C to 42 ° C.

In nucleic acid hybridization reactions, the conditions used to achieve a specific level of stringency will vary depending on the nature of the nucleic acids to be hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (such as GC content relative to AT), and type of nucleic acid (such as RNA or DNA) of the regions can be considered. nucleic acid hybridization by selecting hybridization conditions. A further consideration is whether one of the nucleic acids is immobilized, for example, in a filter.

Hybridization can be carried out under conditions of low stringency, moderate stringency, or high stringency. As an example of nucleic acid hybridization, a polymeric membrane containing denatured nucleic acids immobilized for 30 minutes at 45 ° C in a solution consisting of 0.9M NaCl, 50mM NaH2PO4, pH 7.0.5 is first prehybridized. , 0 mM Na2EDTA, 0.5% SDS, 10 × Denhardt's solution and 0.5 mg / ml polyriboadenylic acid. Approximately 2 × 107 cpm (specific activity 4-9 × 108 cpm / µg) of 32P end-labeled nucleotide probe is then added to the solution. After 12-16 hours incubation, the membrane is washed for 30 minutes at room temperature in 1 × SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0, 5% SDS, with a subsequent 30 minute wash in a new 1 × SET at Tf-10 ° C for the oligonucleotide probe. The membrane is then exposed to an autoradiographic film to detect hybridization signals. All preceding hybridizations would be considered hybridizations under high stringency conditions.

After hybridization, the filter can be washed to remove any specifically detectable unbound probe. The stringency used to wash the filters can also be varied depending on the nature of the nucleic acids to be hybridized, the length of the nucleic acids to be hybridized, the degree of complementarity, the composition of the nucleotide sequence ( such as GC content relative to AT) and the type of nucleic acid (such as RNA or DNA). Following are examples of washes under progressively higher stringency conditions: 2X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes between hybridization temperature and 68 ° C (high stringency); and 0.15M NaCl for 15 minutes at 72 ° C (very high stringency). A low stringency final wash can be carried out in 0.1X SSC at room temperature. The examples above are merely illustrative of a set of conditions that can be used to wash the filters. One of skill in the art will know that there are numerous recipes for different stringent washes. Other examples are provided below.

In some embodiments, the hybridization conditions comprise a washing step comprising washing for 30 minutes at room temperature in a solution comprising 1 × 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM of Na2EDTA, 0.5% SDS, with a subsequent 30 minute wash in fresh solution.

Nucleic acids that hybridized to the probe are identified by autoradiography or other conventional techniques.

The foregoing procedures can be modified to identify nucleic acids that have reduced levels of sequential identity (homology) with the probe sequence. For example, to obtain nucleic acids of reduced sequential identity (homology) relative to the detectable probe, less stringent conditions can be used. For example, the hybridization temperature can be reduced in 5 ° C increments from 68 ° C to 42 ° C in a hybridization buffer solution that has a Na + concentration of approximately 1M. After hybridization, the filter can be washed with 2X SSC, 0.5% SDS at the hybridization temperature. These conditions are considered to be "moderate" conditions above 50 ° C and "low" conditions below 50 ° C. A specific example of "moderate" hybridization conditions is when hybridization is carried out at 55 ° C. A specific example of "low stringency" hybridization conditions is when the above hybridization is carried out at 45 ° C.

Alternatively, hybridization can be carried out in buffers, such as 6X SSC containing formamide at a temperature of 42 ° C. In this case, the concentration of formamide in the solution

Hybridization buffer can be reduced in 5% increments from 50% to 0% to identify clones that have reduced levels of homology with the probe. After hybridization, the filter can be washed with 6X SSC, 0.5% SDS at 50 ° C. These conditions are considered to be "moderate" conditions above 25% formamide and "low" conditions below 25% formamide. A specific example of "moderate" hybridization conditions is when hybridization is carried out at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is carried out at 10% formamide.

However, the selection of a hybridization format may not be essential - the stringency of the wash conditions is what establishes the conditions that determine whether a nucleic acid is within the scope of the disclosure. The washing conditions used to identify nucleic acids within the scope of the invention include, for example: a salt concentration of about 0.15 M NaCl at 72 ° C for about 15 minutes. See Sambrook ed., MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), Volumes 1-3, Cold Spring Harbor Laboratory (1989), LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993) and Ausubel, ed. John Wiley & Sons, Inc., New York (1997) for a description of the SSC buffer solution and equivalent conditions.

These methods can be used to isolate or identify nucleic acids disclosed herein and the sequences substantially identical thereto. For example, the foregoing methods can be used to isolate or identify nucleic acids that have a sequence of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequential identity (homology) with a nucleic acid sequence selected from the group consisting of one of the sequences disclosed herein and sequences substantially identical to the same or fragments comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 consecutive bases thereof and the complementary sequences to them. Sequential identity (homology) can be measured using the alignment algorithm. For example, homologous polynucleotides may have a coding sequence that is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variants can have a substitution, deletion or addition of one or more nucleotides when compared to the nucleic acids disclosed herein. Additionally, the aforementioned procedures can be used to isolate nucleic acids encoding polypeptides having at least about 99%, 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% , at least 65%, at least 60%, at least 55% or at least 50% sequential identity (homology) with a polypeptide disclosed herein or fragments comprising at least 5, 10, 15, 20, 25, 30 , 35, 40, 50, 75, 100 or 150 consecutive amino acids thereof as determined using a sequence alignment algorithm (such as the FASTA version 3.0t78 algorithm with predetermined parameters).

Oligonucleotide probes and methods of using them

The disclosure also provides nucleic acid probes that can be used, for example, to identify, amplify, or isolate nucleic acids that encode a polypeptide that has enzymatic activity of aldolase, such as pyruvate aldolase, such as HMG and / or KHG, or fragments thereof. or to identify aldolase enzyme genes, such as pyruvate aldolase, such as HMG and / or KHG aldolase. In some examples, the probe comprises at least about 10 consecutive bases of a nucleic acid disclosed herein. Alternatively, a probe disclosed herein can have at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or approximately 10 to 50, approximately 20 to 60, approximately 30 to 70 consecutive bases of a sequence as set forth in a nucleic acid disclosed herein. Probes identify a nucleic acid by binding and / or hybridization. Probes can be used in arrays disclosed herein, see description below, including capillary arrays. The probes disclosed herein can further be used to isolate other nucleic acids or polypeptides.

The isolated, synthetic or recombinant nucleic acids disclosed herein, the sequences complementary thereto or a fragment comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200 , 300, 400 or 500 consecutive bases of one of the sequences disclosed herein or sequences complementary thereto can also be used as probes to determine whether a biological sample, such as an oil sample, contains an organism having a sequence nucleic acid disclosed herein or an organism from which nucleic acid was obtained. In such procedures, a biological sample is obtained that is potentially harboring the organism from which the nucleic acid was isolated, and nucleic acids are obtained from the sample. Nucleic acids are contacted with the probe under conditions that allow the probe to specifically hybridize to any sequence complementary to those present therein.

When necessary, conditions that allow the probe to specifically hybridize to complementary sequences can be determined by contacting the probe with complementary sequences from samples that

they are known to contain the complementary sequence as well as control sequences that do not have the complementary sequence. Hybridization conditions, such as the salt concentration of the hybridization buffer, the concentration of formamide in the hybridization buffer, or the hybridization temperature can be varied to identify conditions that allow the probe to hybridize specifically with complementary nucleic acids .

If the sample contains the organism from which the nucleic acid was isolated, then specific probe hybridization is detected. Hybridization can be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye, or an enzyme capable of catalysing the formation of a detectable product.

Many methods are known to those skilled in the art for using the labeled probes to detect the presence of complementary nucleic acids in a sample. These include Southern Blots, Northern Blots, colony hybridization procedures, and blot blots. Protocols for each of these procedures are provided in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2a Ed., Cold Spring Harbor Laboratory Press (1989).

Alternatively, more than one probe (at least one that is capable of specifically hybridizing to any complementary sequence that is present in the nucleic acid sample) can be used in an amplification reaction to determine if the sample contains an organism that contains a nucleic acid sequence disclosed herein (such as an organism from which nucleic acid was isolated). In some embodiments, the probes comprise oligonucleotides. In some embodiments, the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook, supra. Alternatively, the amplification may comprise a ligase chain reaction, 3SR, or a chain displacement reaction. (See Barany, F., "The Ligase Chain Reaction in a PCR World", PCR Methods and Applications 1: 5-16, 1991;

E. Fahy et al., "Self-sustained Sequence Replication (3SR): An Isothermal Transcription-based Amplification System Alternative to PCR", PCR Methods and Applications 1: 25-33, 1991; and Walker G. T. et al., "Strand Displacement Amplification — an Isothermal in vitro DNA Amplification Technique", Nucleic Acid Research 20: 1691-1696, 1992). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is carried out, and any resulting amplification products are detected. The amplification product can be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium bromide. Alternatively, one or more of the probes can be labeled with a radioactive isotope and the presence of a radioactive amplification product can be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences disclosed herein can also be used in chromosome displacement procedures to identify clones containing genomic sequences adjacent to the sequences disclosed herein. Such methods allow the isolation of genes encoding additional proteins from the host organism.

In some embodiments, the isolated, synthetic, or recombinant nucleic acids disclosed herein, the sequences complementary thereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 or more consecutive bases of one of the sequences disclosed herein or sequences complementary thereto are used as probes to identify and isolate related nucleic acids. In some embodiments, the related nucleic acids can be genomic cDNAs or DNAs from organisms other than the organism from which the nucleic acid was isolated. For example, the other organisms may be related organisms. In such procedures, a nucleic acid sample is contacted with the probe under conditions that allow the probe to specifically hybridize to related sequences. Hybridization of the probe with the nucleic acids of the organism listed below is detected using any of the methods described above.

By varying the stringency of the hybridization conditions used to identify nucleic acids, such as cDNAs or genomic DNAs, which hybridize to detectable probes, nucleic acids that have different levels of homology to the probe can be identified and isolated. The stringency can be varied by carrying out the hybridization at temperatures varying below the melting temperatures of the probes. The melting temperature, Tf, is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes with a perfectly complementary probe. Very stringent conditions are selected at about 5 ° C less than or equal to Tf for a specific probe. The melting temperature of the probe can be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length, the fusion temperature (Tf) is calculated using the formula: Tf = 81.5 + 16.6 (log [Na +]) + 0.41 (fraction G + C) - (600 / N) where N is the length of the probe.

If hybridization is carried out in a solution containing formamide, the melting temperature can be calculated using the equation: Tf = 81.5 + 16.6 (log [Na +]) + 0.41 (fraction G + C) - (0.63% formamide) - (600 / N) where N is the length of the probe.

Prehybridization can be performed in 6X SSC, 5 × Denhardt's reagent, 0.5% SDS, 100 µg / ml fragmented denatured salmon sperm DNA, or 6X SSC, 5 × Denhardt's reagent, 0, 5% SDS, 100 µg / ml fragmented denatured salmon sperm DNA, 50% formamide. The formulas for the SSC and Denhardt solutions are indicated in Sambrook et al., Supra.

In some embodiments, hybridization is accomplished by adding the detectable probe into the prehybridization solutions noted above. When the probe comprises double-stranded DNA, it is denatured before the addition of the hybridization solution. In some embodiments, the filter is contacted with the hybridization solution for a period of time sufficient to allow the probe to hybridize to genomic cDNAs or DNAs containing sequences complementary to or homologous to it. For probes over 200 nucleotides in length, hybridization can be carried out at 15-25 ° C below Tf. For shorter probes, such oligonucleotide probes, hybridization can be carried out at 5-10 ° C below Tf. In some embodiments, for 6X SSC hybridizations, hybridization is carried out at approximately 68 ° C. Generally, for hybridizations in solutions containing 50% formamide, hybridization is carried out at approximately 42 ° C.

Inhibition of the expression of aldolase enzymes

The disclosure provides complementary nucleic acids (such as antisense sequences) to the nucleic acids disclosed herein, such as nucleic acids encoding aldolase enzymes, such as nucleic acids comprising antisense, siRNAs, miRNAs, and ribozymes. The nucleic acids disclosed herein that comprise antisense sequences may be capable of inhibiting the transport, splicing, or transcription of genes encoding aldolase enzymes. Inhibition can be accomplished through targeting of genomic DNA or messenger RNA. Transcription or function of the target nucleic acid can be inhibited, for example, by hybridization and / or cleavage. In an exemplary set of inhibitors provided by the present invention includes oligonucleotides that are capable of binding to the aldolase enzyme gene or message, such as pyruvate aldolase, such as HMG and / or KHG aldolase, in both cases preventing or inhibiting the production or function of an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase. Association can be through sequence specific hybridization. Another type of useful inhibitors includes oligonucleotides that cause inactivation or cleavage of the message of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The oligonucleotide can have enzymatic activity which causes such cleavage, such as ribozymes. The oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. A pool of several such different oligonucleotides can be screened for those with the desired activity. Accordingly, the disclosure provides various compositions for the inhibition of aldolase enzyme expression, such as pyruvate aldolase, such as HMG and / or KHG aldolase at the nucleic acid and / or protein level, such as antisense, siRNA, miRNA and ribozymes comprising aldolase enzyme sequences, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein, and anti-aldolase antibodies, such as anti-pyruvate aldolase, such as anti-HMG and / or anti -KHG aldolase disclosed herein.

Inhibition of the expression of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase can have a variety of industrial applications. For example, inhibition of the expression of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase can slow or prevent spoilage of food. In some embodiments, the use of compositions disclosed herein that inhibit the expression and / or activity of aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase, such as antibodies, antisense oligonucleotides, ribozymes, siRNAs, and miRNAs are used to slow or prevent spoilage of food. Accordingly, in some embodiments, methods and compositions are disclosed which comprise the application on a plant or plant product (such as a cereal, a grain, fruit, seed, root, leaf, etc.) of antibodies, antisense oligonucleotides, ribozymes, siRNAs. and miRNAs disclosed herein to slow or prevent spoilage of food. These compositions can also be expressed by the plant (such as a transgenic plant) or other organism (such as a bacterium or other microorganism transformed with an aldolase enzyme gene, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed in the Present).

The compositions disclosed herein for the inhibition of aldolase enzyme expression, such as pyruvate aldolase, such as HMG and / or KHG aldolase (such as RNAi, antisense ribozymes, antibodies) can be used as pharmaceutical compositions, such as antipathogenic agents. or in other therapies, such as antimicrobial agents for, for example, Salmonella.

Antisense oligonucleotides

The disclosure provides antisense oligonucleotides capable of binding to an enzyme aldolase message, such as pyruvate aldolase, such as HMG and / or KHG aldolase, which, in some embodiments, can inhibit the enzyme activity of the enzyme aldolase, such as pyruvate aldolase , such as HMG and / or KHG aldolase targeting mRNA. Strategies for designing oligonucleotides are described in the scientific and patent literature, and those aldolase enzyme oligonucleotides, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be designed by the person skilled in the art using the new reagents disclosed herein. For example, the protocols of

gene shuffling / RNA mapping to detect effective antisense oligonucleotides are known in the art, see Ho (2000) Methods Enzymol. 314: 168-183, which describes an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for convincing selection of antisense sequences. See also Smith (2000) Eur. J. Pharm. Sci. 11: 191-198.

Naturally occurring nucleic acids are used as antisense oligonucleotides. These antisense oligonucleotides can be any length; for example, in other embodiments, antisense oligonucleotides are from about 5 to about 100, about 10 to about 80, about 15 to about 60, about 18 to about 40. The optimal length can be determined by routine detection. Antisense oligonucleotides can be present in any concentration. The optimal concentration can be determined by routine detection. A wide variety of synthetic, unnaturally derived nucleotides and nucleic acid analogs are known that can treat this potential problem. For example, peptide nucleic acids (PNAs) containing a nonionic backbone can be used, such as N- (2-aminoethyl) glycine units. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144: 189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogs provided in the invention may also include phosphor dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulphamate, 3'-thioacetal, methylene (methylimino), 3'-N- nucleic acids. carbamate and morpholino carbamate, such as those described above.

Combinatorial chemistry methodology can be used to create large amounts of oligonucleotides that can be quickly screened for specific oligonucleotides that have the appropriate binding affinities and specificities for any target, such as aldolase enzyme sequences, such as pyruvate aldolase, such as HMG and / or KHG sense and antisense aldolase disclosed herein (see Gold (1995) J. of Biol. Chem. 270: 13581-13584).

Inhibitory ribozymes

The invention provides ribozymes capable of binding the enzyme aldolase message, such as pyruvate aldolase, such as HMG and / or KHG aldolase. These ribozymes can inhibit aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, for example, by targeting mRNA. Strategies for designing ribozymes and selecting the enzyme aldolase-specific antisense sequence, such as pyruvate aldolase, such as HMG and / or KHG aldolase as the target, are well described in the scientific and patent literature, and those skilled in the art can design such ribozymes using the novel reagents disclosed herein. Ribozymes work by binding to a target RNA through the RNA-binding portion of a ribozyme that is maintained in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds to a target RNA through complementary base pairing, and once attached to the correct site, it acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in this way will destroy its ability to direct the synthesis of an encoded protein if cleavage occurs in the encoding sequence. After a ribozyme has bound and cleaved from its RNA target, it can break free of that RNA to repeatedly bind and cleave new targets.

In some circumstances, the enzymatic nature of a ribozyme may be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation, or association with another molecule) such as the effective concentration of the ribozyme necessary to carry out a therapeutic treatment may be less than that of an antisense oligonucleotide. This potential advantage reflects the ribozyme's ability to act enzymatically. Therefore, a single ribozyme is capable of cleaving various target RNA molecules. In some embodiments, a ribozyme is a highly specific inhibitor, with the specificity of inhibition that depends not only on the binding base pairing mechanism, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target, and therefore specificity is defined as the ratio of the cleavage rate of targeted RNA to the cleavage rate of non-targeted RNA. This cleavage mechanism depends on factors additional to those involved in base pairing. Therefore, the specificity of the action of a ribozyme may be higher than that of the antisense oligonucleotide that binds to the same RNA site.

The ribozyme disclosed herein such as an enzymatic ribozyme RNA molecule can be formed into a hammerhead motif, hairpin motif, a group I intron motif and / or a RNasaP-like RNA in association with a sequence of RNA guide. Examples of hammerhead motifs are described, for example, by Rossi (1992) Aids Research and Human Retroviruses 8: 183; hairpin motifs by Hampel (1989) Biochemistry 28: 4929, and Hampel (1990) Nuc. Acids Res. 18: 299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry 31:16; the RNasaP motif by Guerrier-Takada (1983) Cell 35: 849; and the group I intron by Cech, US Patent No. 4,987,071. Those skilled in the art will recognize that a ribozyme disclosed herein, such as an enzymatic RNA molecule of the present invention, may have a specific substrate binding site complementary to one or more of the RNA regions of the target gene. A

Ribozyme disclosed herein can have a nucleotide sequence within or around the substrate binding site that imparts RNA cleavage activity to the molecule.

RNA interference (RNAi)

In some embodiments, the disclosure provides RNA inhibitory molecules, so-called "RNAi" molecules, which comprise aldolase enzyme sequences, such as pyruvate aldolase, HMG and / or KHG aldolase, disclosed herein. The RNAi molecule may comprise a double-stranded RNA molecule (dsRNA), such as siRNA, miRNA and / or short hairpin RNA (shRNA) molecules. The RNAi molecule, such as siRNA (small inhibitory RNA) and / or miRNA (microRNA), can inhibit the expression of an aldolase enzyme gene, such as pyruvate aldolase, HMG and / or KHG aldolase. In some embodiments the RNAi molecule, such as siRNA and / or miRNA is approximately 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more duplex nucleotides in length. While the disclosure is not limited to any particular mechanism of action, RNAi can enter a cell and cause degradation of single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), the mRNA of the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi is the breakage of a double-stranded RNA (dsRNA) that matches a specific gene sequence into short pieces called small interfering RNAs, which triggers the degradation of the mRNA that matches its sequence. In some embodiments, the RNAi disclosed herein are used in gene silencing treatments, see Shuey (2002) Drug Discov. Today 7: 1040-1046. In some embodiments the disclosure provides methods for selectively degrading RNA using RNAi molecules such as siRNA and / or miRNA. The process can be carried out in vitro, ex vivo or in vivo. In some embodiments, the RNAi molecules disclosed herein can be used to generate a loss-of-function mutation in a cell, organ, or animal.

In one aspect, intracellular introduction of RNAi occurs by internalization of a target cell-specific ligand bound to an RNA binding protein comprising an RNAi (such as microRNA) that is adsorbed. The ligand is specific to a single target cell surface antigen. The ligand can spontaneously internalize after binding to the cell surface antigen. If the single cell surface antigen does not naturally internalize after binding to its ligand, internalization can be promoted by incorporating an arginine-rich peptide or another membrane-permeable peptide into the ligand structure or the RNA-binding protein or binding of said peptide to the ligand or RNA binding protein. See United States Patent Application No. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the disclosure provides lipid-based formulations for administration, such as introducing nucleic acids disclosed herein as lipid-nucleic acid particles comprising an RNAi molecule into a cell, see for example, Application Publication U.S. Patent No. 20060008910.

Nucleic acid modification - preparation of variant enzymes

Methods for generating variants of the nucleic acids disclosed herein are disclosed, such as those encoding an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase. These methods can be repeated or used in various combinations to generate aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase that have altered or different activity or altered or different stability of an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase encoded by the template nucleic acid. These methods can also be repeated or used in various combinations such as to generate variations in gene / message expression, message translation, or message stability. In other embodiments, the genetic makeup of a cell is altered by, for example, modification of a homologous gene ex vivo, followed by its reinsertion into the cell.

A nucleic acid disclosed herein can be altered in several ways. For example, random or stochastic, or non-stochastic, or "directed evolution" methods, see US Patent No.

6,361,974. Methods for random gene mutation are well known in the art, see US Patent No. 5,830,696. For example, mutagens can be used to randomly mutate a gene. Mutagens include, for example, ultraviolet light or gamma irradiation, or a chemical mutagen, such as mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks to be repaired by recombination. Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine, or formic acid. Other mutagens are nucleotide precursor analogs, such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Interleaving agents such as proflavin, acriflavin, quinacrine and the like can also be used.

Any molecular biology technique can be used, such as random PCR mutagenesis, see Rice (1992) Proc. Natl. Acad. Sci. United States 89: 5467-5471; or combinatorial multiple cassette mutagenesis, see Crameri (1995) Biotechniques 18: 194-196. Alternatively, nucleic acids, such as genes, can then be brought together

for random or "stochastic" fragmentation, see US Patent No. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. In other embodiments, modifications, additions, or deletions are introduced by error-prone PCR, transposition, cassette mutagenesis, recursive assembly mutagenesis, exponential assembly mutagenesis, site-specific mutagenesis, gene reassembly (such as gene reassembly). , see US Patent No. 6,537,776), gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, template mutagenesis containing uracil, gap duplex mutagenesis, point match repair mutagenesis, poor host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction mutagenesis purification, artificial gene synthesis, mutagenesis of assembly, creation of chimeric nucleic acid multimers or, chromosomal saturation mutagenesis (CSM) and / or combination of these and other methods.

The following publications describe a variety of recursive recombination procedures and / or methods that can be incorporated into the methods disclosed herein: Stemmer (1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4: 1-4; Ness (1999) Nature Biotechnology 17: 893-896; Chang (1999) "Evolution of a cytokine using DNA family shuffling" Nature Biotechnology 17: 793-797; Minshull (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3: 284-290; Christians (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17: 259-264; Crameri (1998) “DNA shuffling of a family of genes from diverse species accelerates directed evolution” Nature 391: 288-291; Crameri (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology 15: 436-438; Zhang (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci. United States 94: 4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8: 724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling" Nature Medicine 2: 100-103; Gates et al. (1996) "Affinity selective isolation of ligands from peptide libraries through display on a lac repressor headpiece dimer" Journal of Molecular Biology 255: 373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" in: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18: 194-195; Stemmer et al. (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxyribonucleotides" Gene, 164: 49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio / Technology 13: 549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370: 389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution." Proc. Natl. Acad. Sci. United States 91: 10747-10751.

Mutation methods to generate diversity include, for example, site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254 (2): 157-178; Dale et al. ( 1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods Mol. Biol. 57: 369-374; Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19: 423-462; Botstein & Shortle (1985 ) "Strategies and applications of in vitro mutagenesis" Science 229: 1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237: 1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, DMJ eds., Springer Verlag, Berlin)); mutagenesis using templates containing uracil (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection "Methods in Enzymol. 154, 367-382; and Bass et al. (1988)" Mutant Trp repressors with new DNAbinding specificities "Science 242: 240-245); oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment "Nucleic Acids Res. 10: 6487-6500; Zoller & Smith (1983)" Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors "Methods in Enzymol. 100: 468-500; and Zoller (1987) Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template ”Methods in Enzymol. 154: 329-350); phosphorothioate-modified DNA mutagenesis (Taylor (1985) “The use of phosphorothioatemodified DNA in restriction enzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor (1985) “The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA ”Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye (1986)“ Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis ”Nucl. Acids Res. 14: 96799698; Sayers (1988) "YT Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16: 791-802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide ”Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol. " Oligonucleotide-directed construction of mutations via gapped duplex DNA ”154: 350-367; Kramer (1988)“ Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction

of mutations ”Nucl. Acids Res. 16: 7207; and Fritz (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the method include mismatch repair (Kramer (1984) "Point Mismatch Repair" Cell 38: 879-887), mutagenesis using repair deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors" Methods in Enzymol. 154: 382-403), mutagenesis of deletion (Eghtedarzadeh (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res. 14: 5115), restriction-selection and restriction-selection and restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin "Phil. Trans.

R. Soc. Lond. A 317: 415-423), total gene synthesis mutagenesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transduction)" Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites ”Gene 34: 315-323; and Grundstrom et al. (1985)“ Oligonucleotidedirected mutagenesis by microscale 'shot-gun' gene synthesis ”Nucl. Acids Res. 13: 3305-3316) , double strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4: 450-455. "Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis ”Proc. Natl. Acad. Sci. USA, 83: 7177-7181). Additional details on any of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for troubleshooting problems with various methods of mutagenesis.

Protocols that can be used to practice the method are described, for example, in US Patent No. 5,605,793 to Stemmer (Feb. 25, 1997), "" Methods for In Vitro Recombination "; United States Patent No. 5,811,238 to Stemmer et al. (September 22, 1998) "Methods to generate polynucleotides that have desired characteristics by iterative selection and recombination"; United States Patent No. 5,830,721 to Stemmer et al. (Nov 3, 1998), "DNA Mutagenesis by Random Fragmentation and Reassembly"; United States Patent No. 5,834,252 to Stemmer, et al. (Nov 10, 1998) "Final Complementary Polymerase Reaction"; United States Patent No. 5,837,458 to Minshull, et al. (Nov 17, 1998), “Methods and Compositions for Cellular and Metabolic Manipulation”; WO 95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and Reassembly"; WO 96/33207 to Stemmer and Lipschutz "Final Complementary Polymerase Chain Reaction"; WO 97/20078 to Stemmer and Crameri "Methods for generating polynucleotides having desired characteristics by selection and iterative recombination"; WO 97/35966 to Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Manipulation"; WO 99/41402 to Punnonen et al. "Targeting of Genetic Vaccine Vectors"; WO 99/41383 to Punnonen et al. "Antigen Library Immunization"; WO 99/41369 to Punnonen et al. "Manipulation of genetic vaccine vector"; WO 99/41368 to Punnonen et al. "Optimization of immunomodulatory properties of genetic vaccines"; EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly"; EP 0932670 from Stemmer "Evolution of cellular DNA uptake by recursive sequence recombination"; WO 99/23107 by Stemmer et al., "Modification of virus tropism and host rank by transposition of the viral genome"; WO 99/21979 to Apt et al., "Human Papillomavirus Vectors"; WO 98/31837 of del Cardayre et al. "Evolution of whole cells and organisms by recursive sequence recombination"; WO 98/27230 to Patten and Stemmer, "Methods and Compositions for Manipulating Polypeptides"; WO 98/27230 to Stemmer et al., "Methods for optimization of gene therapy by transposition and recursive sequence selection," WO 00/00632, "Methods for generating highly diverse libraries"; WO 00/09679, "Methods for Obtaining Banks and Resulting Sequences of Recombinant Polynucleotides in Vitro"; WO 98/42832 to Arnold et al., "Recombination of polynucleotide sequences using random or defined primers"; WO 99/29902 to Arnold et al., "Method for creating polynucleotide and polypeptide sequences"; WO 98/41653 to Vind, "An in vitro method for constructing a DNA library"; WO 98/41622 by Borchert et al., "Method for constructing a library using DNA rearrangement," and WO 98/42727 by Pati and Zarling, "Sequence alterations using homologous recombination."

Protocols that can be used to practice the method (providing details regarding various methods of generating diversity) are described, for example, in US Patent Application Serial No. 09 / 407,800, "TRANSPOSITION OF GENES ALTERED BY CODONS ”by Patten et al. presented on September 28. from 1999; "EVOLUTION OF CELLS AND WHOLE ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" de del Cardayre et al., US Patent No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., US Patent No. 6,319,714; 6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT / US00 / 01203; "USE OF VARIED OLIGONUCLEOTIDE SYNTHESIS OF CODONS FOR SYNTHETIC TRANSPOSITION" by Welch et al., US Patent No. 6,436,675; Selifonov et al. "METHODS FOR MAKING CHARACTER, POLYNUCLEOTID AND POLYPEPTIDE CHAINS WITH DESIRED CHARACTERISTICS", presented on Jan. 18. 2000, (PCT / US00 / 01202) and, for example, “METHODS FOR CONDUCTING CHAINS OF CHARACTERS, POLYNUCLEOTIDES AND POLYPEPTIDES WITH DESIRED CHARACTERISTICS” by Selifonov et al., presented on Jul. 18. 2000 (US Serial No. 09 / 618,579); "METHODS FOR POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer,

submitted on Jan 18. 2000 (PCT / US00 / 01138); and Affholter's “MEDICALLY MEDICALLY RECOMBINED ON NUCLEIC ACID NUCLEIC ACID FRAGMENTS AND INSULATION”, presented on September 6. 2000 (US Serial No. 09 / 656,549); and United States Patents No. 6,177,263; 6,153,410.

Non-stochastic or "directed evolution" methods include, for example, saturation mutagenesis, such as gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the Nucleic acids disclosed herein to generate aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase with new or altered properties (such as activity under highly acidic or alkaline conditions, high or low temperatures, and the like). Polypeptides encoded by the modified nucleic acids can be tested for activity before being evaluated for cleavage formation or other activity. Any evaluation modality or protocol can be used such as using a capillary fixation platform. See United States Patent No. 6,361,974; 6,280,926;

5,939,250.

Gene site saturation mutagenesis or GSSM

Methods for making enzyme using gene site saturation mutagenesis or GSSM are further described, as described herein, and also in US Patent No. 6,171,820 and 6,579,258. In some embodiments, codon primers containing a degenerate N, N, G / T sequence are used to introduce point mutations into a polynucleotide such as an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase or a disclosed antibody. herein in order to generate a set of progeny polypeptides representing a full range of unique amino acid substitutions at each amino acid position, such as an amino acid residue at an active enzyme site or ligand binding site directed to modify. These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N, N, G / T sequence and optionally a second homologous sequence. Downstream translational progeny products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site throughout the polypeptide because N, N, G / T sequence degeneracy includes codons for all 20 amino acids . In some embodiments, a degenerate oligonucleotide (comprised of a degenerate N, N, G / T cassette) is used to subject each original codon in a parent polynucleotide template to a full range of codon substitutions. In other embodiments, at least two degenerate cassettes are used - whether in the same oligonucleotide or not, to subject at least two original codons in a parent polynucleotide template to a full range of codon substitutions. For example, more than one N, N, G / T sequence may be contained in an oligonucleotide to introduce amino acid mutations at more than one site. This plurality of N, N, G / T sequences can be directly contiguous or separated by one or more additional nucleotide sequences. In other embodiments, oligonucleotides useful for introducing additions and deletions can be used alone or in combination with codons that contain an N, N, G / T sequence to introduce any combination or permutation of amino acid additions, deletions, and / or substitutions.

In some embodiments, simultaneous mutagenesis of two or more contiguous amino acid positions is performed using an oligonucleotide containing contiguous N, N, G / T triplets, ie, a degenerate (N, N, G / T) n sequence. In other embodiments, degenerate cassettes are used that have less degeneration than the N, N, G / T sequence. For example, it may be desired in some cases that a degenerate triplet sequence composed of only one N is used (such as in an oligonucleotide), where said N may be in the first, second, or third position of the triplet. Any other base that includes any combination and permutation of the same can be used in the remaining two positions of the triplet. Alternatively, it may be desired in some cases to use (such as in an oligo) a degenerate N, N, N triplet sequence.

In some embodiments, the use of degenerate triplets (such as N, N, G / T triplets) allows for a systematic and easy generation of a full range of natural amino acids (for a total of 20 amino acids) at each and every position of amino acids in a polypeptide (in other embodiments, the methods also include generating fewer than all possible substitutions for amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 different species can be generated (ie 20 possible amino acids per position X 100 amino acid positions). Through the use of an oligonucleotide or set of oligonucleotides containing a degenerate N, N, G / T triplet, 32 individual sequences can encode 20 possible natural amino acids. Therefore, in a reaction vessel in which a parent polynucleotide sequence is subjected to saturation mutagenesis using at least one of said oligonucleotide, 32 different progeny polynucleotides encoding 20 different polypeptides are generated. In contrast, the use of a non-degenerate oligonucleotide in site directed mutagenesis leads to only one progeny polypeptide product per reaction vessel. Non-degenerate oligonucleotides can optionally be used in combination with disclosed degenerate primers; for example, non-degenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides a means of generating specific silent point mutations, point mutations that lead to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.

In some embodiments, each saturation mutagenesis reaction vessel contains polynucleotides that encode at least 20 progeny polypeptide molecules (such as an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase) such that all the 20 natural amino acids are represented at a specific amino acid position that corresponds to the mutagenized codon position in the parent polynucleotide (other embodiments use less than 20 natural combinations). The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subject to clonal amplification (such as cloned into a suitable host, such as E. coli host using for example an expression vector) and undergo expression screening. When an individual progeny polypeptide is identified by detection to show a favorable change in property (when compared to the parent polypeptide, such as increased carbon-carbon formation or cleavage activity under alkaline or acidic conditions), it can be sequenced to identify the favorable amino acid substitution contained in it.

In some embodiments, after mutagenizing each and every amino acid position in a parent polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes can be identified in more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or parts of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified at each of the 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Therefore, there are 3 × 3 × 3 or 27 total possibilities, including 7 that were previously examined - 6 unique point mutations (i.e. 2 at each of the three positions) and unchanged at any position.

In yet another embodiment, site saturation mutagenesis can be used in conjunction with rearrangement, chimerization, recombination, and other mutagenesis processes, along with detection. This invention provides the use of any mutagenesis process, including saturation mutagenesis, in an iterative way. In one example, the iterative use of any mutagenesis process is used in combination with detection.

Also disclosed is the use of proprietary codon primers (containing a degenerate N, N, N sequence) to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides representing a full range of substitutions. of unique amino acids at each amino acid position (gene site saturation mutagenesis (GSSM)). The oligos used are contiguously composed of a first homologous sequence, a degenerate N, N, N sequence and in some embodiments but not necessarily a second homologous sequence. The downstream progeny translational products of the use of such oligonucleotides include all possible amino acid changes at each amino acid site throughout the polypeptide because N, N, N sequence degeneracy includes codons for all 20 amino acids.

In some embodiments, a degenerate oligo (consisting of a degenerate N, N, N cassette) is used to subject each original codon in a parent polynucleotide template to a full range of codon substitutions. In other embodiments, at least two degenerate N, N, N cassettes are used - whether in the same oligonucleotide or not, to subject at least two original codons in a parent polynucleotide template to a full range of codon substitutions. Therefore, more than one N, N, N sequence may be contained in an oligo to introduce amino acid mutations at more than one site. This plurality of N, N, N sequences can be directly contiguous or separated by one or more additional nucleotide sequences. This plurality of N, N, N sequences can be directly contiguous or separated by one or more additional nucleotide sequences. In other embodiments, useful oligos for introducing additions and deletions can be used alone or in combination with codons containing an N, N, N sequence to introduce any combination or permutation of additions, deletions and / or amino acid substitutions.

In some embodiments, it is possible to simultaneously mutagenize two or more contiguous amino acid positions using an oligonucleotide containing contiguous N, N, N triplets, ie, a degenerate (N, N, N) n sequence. The use of degenerate cassettes that have less degeneration than the N, N, N sequence is also described. For example, in some cases it may be desired to use (such as in an oligo) a degenerate triplet sequence consisting of only one N, where said N may be in the first, second, or third position of the triplet. Any other base that includes any combination and permutation of the same can be used in the remaining two positions of the triplet. Alternatively, it may be desired in some cases to use (such as in an oligo) a degenerate N, N, N triplet sequence, an N, N, G / T or N, N, G / C triplet sequence.

In some embodiments, the use of a degenerate triplet (such as an N, N, G / T, or N, N, G / C triplet sequence) is advantageous for several reasons. This disclosure provides means for systematically and relatively easily generating the replacement of the full range of possible amino acids (for a total of 20 amino acids) at each and every amino acid position in a polypeptide. Therefore, for a 100 amino acid polypeptide, the disclosure provides a means for systematically and relatively easily generating 2000 distinct species (ie, 20 possible amino acids per 100 amino acid positions). It is appreciated that it is provided, through the use of an oligo containing a triplet sequence N, N, G / T or N, N, G / C

degenerate, 32 individual sequences encoding 20 possible amino acids. Therefore, in a reaction vessel in which a parent polynucleotide sequence is subjected to saturation mutagenesis using at least one of said oligo, 32 different progeny polynucleotides encoding 20 different polypeptides are generated. In contrast, the use of a non-degenerate oligo in site directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.

The use of non-degenerate oligos is also described, which can optionally be used in combination with disclosed degenerate primers. It is appreciated that in some situations, it is advantageous to use non-degenerate oligos to generate specific point mutations in a working polynucleotide. This provides a means of generating specific silent point mutations, point mutations that lead to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.

Therefore, in some embodiments, each saturation mutagenesis reaction vessel contains polynucleotides that encode at least 20 progeny polypeptide molecules so that all 20 amino acids are represented at a specific amino acid position corresponding to the codon position. mutagenized in the parent polynucleotide. 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (such as cloned into a suitable E. coli host using an expression vector) and subjected to detection of expression. When an individual progeny polypeptide is identified by detecting to show a favorable change in property (when compared to the parent polypeptide) it can be sequenced to identify the corresponding favorable amino acid substitution contained therein.

In some embodiments, after mutagenizing each and every amino acid position in a parent polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes can be identified in more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or parts of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified at each of the 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Therefore, there are 3 × 3 × 3 or 27 total possibilities, including 7 that were previously examined - 6 unique point mutations (i.e. 2 at each of the three positions) and unchanged at any position.

Also described is the use of saturation mutagenesis in combination with additional mutagenization processes, such as a process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive rearrangement.

In addition to performing mutagenesis throughout the entire sequence of a gene, the present invention provides that mutagenesis can be used to replace each of any number of bases in a polynucleotide sequence, where the number of bases to be mutagenized is, in some embodiments, any integer from 15 to 100,000. Therefore, instead of mutagenizing each position across a molecule, each can be subjected

or a discrete number of bases (in some embodiments a subset having in its entirety from 15 to 100,000) for mutagenesis. In some embodiments, a separate nucleotide is used to mutagenize each position or group of positions along a polynucleotide sequence. A group of 3 positions to be mutagenized can be a codon. Mutations can be introduced using a mutagenic primer, which contains a heterologous cassette, also called a mutagenic cassette. Examples of cassettes can have from 1 to 500 bases. Each nucleotide position in said heterologous cassettes is N, A, C, G, T, A / C, A / G, A / T, C / G, C / T, G / T, C / G / T, A / G / T, A / C / T, A / C / G or E, where E is any base that is not A, C, G, or T (E may refer to as a designer oligo).

In some embodiments, saturation mutagenesis is comprised of the mutagenesis of a complete set of mutagenic cassettes (where each cassette is, in some embodiments, approximately 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (in where the sequence to be mutagenized is, in some embodiments, approximately 15 to 100,000 bases in length). Therefore, a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to mutagenize. A cluster of mutations to be introduced into a cassette may be different or the same as a second cluster of mutations to be introduced into a second cassette during the application of a round of saturation mutagenesis. Such clusters are exemplified by deletions, additions, clusters of particular codons, and clusters of particular nucleotide cassettes.

In some embodiments, the defined sequences to be mutagenized include an entire gene, pathway, cDNA, an open reading frame (ORF), and an entire promoter, enhancer, repressor / transactivator, origin of replication, intron, operator, or any functional group of polynucleotides. . Generally, a "defined sequence" for this purpose can be any polynucleotide that has a 15 base polynucleotide sequence and polynucleotide sequences of lengths between 15 bases and 15000 bases (this invention specifically names each integer

In between). Considerations for choosing codon groups include amino acid types encoded by a degenerate mutagenic cassette.

In some embodiments, a grouping of mutations that can be introduced into a mutagenic cassette, this invention specifically provides degenerate codon substitutions (using degenerate oligos) encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position and a library of polypeptides encoded thereby.

Synthetic Ligation Reassembly (SLR)

Also described is a non-stochastic gene modification system called "synthetic ligation reassembly" or simply "SLR", a "directed evolution process" to generate polypeptides, such as enzymes or aldolase antibodies, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, with new or altered properties.

SLR is a method of ligation of oligonucleotide fragments non-stochastically. This method differs from stochastic oligonucleotide rearrangement in that the nucleic acid building blocks are not randomly transposed, concatenated, or chimerized, but assembled non-stochastically. See United States Patent No. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776. In some embodiments, the SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to be reassembled in intersection with the template polynucleotide in a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over is reassembled with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology between polynucleotides to rearrange itself. Therefore, this method can be used to generate non-stochastically libraries (or pools) of progeny molecules composed of more than 10,100 different chimeras. The SLR can be used to generate libraries made up of more than 101,000 different chimeras. Therefore, embodiments include non-stochastic methods of producing a finished chimeric nucleic acid molecule set with a general assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having mutually compatible linkable ends and assembling these nucleic acid building blocks in such a way that a general designed assembly order is achieved.

The mutually compatible linkable ends of the nucleic acid building blocks to be assembled are considered "useful" for this type of ordered assembly if they allow the building blocks to mate in predetermined orders. Therefore, the general order of assembly in which the nucleic acid building blocks can be coupled is specified by the design of the linkable ends. If more than one assembly step is used, then the general assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly steps. In some embodiments, the paired building pieces are treated with an enzyme, such as a ligase (such as T4 DNA ligase) to achieve covalent binding of the building pieces.

The mutually compatible linkable ends of the nucleic acid building blocks to be assembled are considered "useful" for this type of ordered assembly if they allow the building blocks to mate in predetermined orders. These parental oligonucleotide templates therefore serve as a source of sequence information that aids in the design of the nucleic acid building blocks to be mutagenized, such as chimerized or transposed. In some embodiments of this method, the sequences of a plurality of parental nucleic acid templates are aligned so as to select one or more demarcation points. The demarcation points may be located in a homology area and are composed of one or more nucleotides. These demarcation points In some embodiments they are shared by at least two of the parent templates. The demarcation points can thus be used to delineate the boundaries of the oligonucleotide building blocks to be generated so as to rearrange the parent polynucleotides. The identified and selected demarcation points on the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules. A demarcation point can be an area of homology (consisting of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences. Alternatively, a demarcation point can be an area of homology that is shared by at least half of the parent polynucleotide sequences, or it can be an area of homology that is shared by at least two thirds of the parent polynucleotide sequences. Furthermore, in some embodiments, useful demarcation points are an area of homology that is shared by at least three-fourths of the parent polynucleotide sequences or may be shared by almost all of the parent polynucleotide sequences. In some embodiments, a demarcation point is an area of homology that is shared by all parental polynucleotide sequences.

In some embodiments, a ligation reassembly process is performed extensively to generate a comprehensive library of progeny chimeric polynucleotides. In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the finished chimeric nucleic acid molecule set. At the same time, in other embodiments, the order of assembly (i.e. the order of assembly of each building block in the 5 'or 3 sequence of each terminated chimeric nucleic acid) in each combination is by design (or non-stochastic) as previously described. Given the non-stochastic nature of the present invention, the possibility of unwanted derivative products is greatly reduced.

In other embodiments, the ligation reassembly method is systematically performed. For example, the method is performed so as to generate a systematically compartmentalized library of progeny molecules, with compartments that can be systematically analyzed, such as one by one. Through the selective and judicious use of specific nucleic acid building blocks coupled with the selective use and judgment of sequentially staggered assembly reactions, a design can be achieved where specific sets of progeny products are made in each of the various reaction vessels. This allows for a systematic examination and screening procedure. Therefore, these methods allow a large number of progeny molecules to be systematically examined in smaller groups. Given their ability to perform chimerizations in a way that is highly flexible but comprehensive and also systematic, particularly when there is a low level of homology between the parent molecules, these methods provide the generation of a library (or set) composed of a large number of progeny molecules. Given the non-stochastic nature of the present ligation reassembly invention, the progeny molecules generated in some embodiments comprise a library of finished chimerized nucleic acid molecules having a general assembly order which is chosen by design. Optimized directed evolution and saturation mutagenesis methods can also be used to generate different molecular species of progeny. It is appreciated that the disclosure provides freedom of choice and control with respect to selection of demarcation points, size and number of nucleic acid building blocks, and size and design of couplings. It is also appreciated that the requirement for intermolecular homology is highly relaxed for the operability of the present invention. In fact, demarcation points can even be chosen in areas of small intermolecular homology or non-homology. For example, due to codon bulking, ie codon degeneration, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding parent template. Alternatively, a codon can be altered such that the coding for an original amino acid is altered. Such substitutions can be introduced into the nucleic acid building block to improve the incidence of homologous intermolecular demarcation points and thus allow an increased number of couplings to be achieved between the building blocks, which in turn allows a greater number of chimeric progeny molecules.

Synthetic gene reassembly

A non-stochastic method called synthetic gene reassembly is also disclosed, which is somehow related to stochastic rearrangement, except that the nucleic acid building blocks are not transposed

or concatenate or chimerize randomly, but are assembled non-stochastically. See United States Patent No. 6,537,776.

The synthetic gene reassembly method does not depend on the presence of a high level of homology between polynucleotides to be transposed. The method can be used to generate non-stochastically libraries (or pools) of progeny molecules composed of more than 10,100 different chimeras. Possibly, the synthetic gene reassembly can still be used to generate libraries made up of more than 101,000 different progeny chimeras.

Therefore, a non-stochastic method of producing a finished chimeric nucleic acid molecule set is disclosed having a general assembly order which is chosen by design, which method is comprised of the steps of generating by design a plurality of blocks of specific nucleic acid construction having mutually useful compatible linkable ends and assembling these nucleic acid building blocks such that a designed overall assembly order is achieved.

The mutually compatible linkable ends of the nucleic acid building blocks to be assembled are considered "useful" for this type of ordered assembly if they allow the building blocks to mate in predetermined orders. Therefore, in some embodiments, the general assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and, if more than one assembly step is used, then the order of General assembly in which nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly steps. In one embodiment, the paired building pieces are treated with an enzyme, such as a ligase (such as T4 DNA ligase) to achieve covalent binding of the building pieces.

In another embodiment, the nucleic acid building block design is obtained after analysis of the sequences of a set of parent nucleic acid templates that serves as the basis for producing a finished set of chimeric nucleic acid molecules. These parent nucleic acid templates therefore serve as a source of sequence information that aids in the design of the nucleic acid building blocks to be mutagenized, i.e. chimerized or transposed.

In one example, the disclosure provides for the chimerization of a family of related genes and their encoded family of related products. In a particular example, the encoded products are enzymes. The aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein can be mutagenized according to the methods described herein.

Therefore, according to one embodiment, the sequences of a plurality of progenitor nucleic acid templates (such as polynucleotides according to the invention) are aligned in order to select one or more demarcation points, which can be located in a homology area. The demarcation points can be used to delineate the boundaries of the nucleic acid building blocks to be generated. Therefore, the identified and selected demarcation points on the progenitor molecules serve as potential chimerization points in the assembly of the progeny molecules.

In some embodiments, a useful demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parent templates, but the demarcation point may be an area of homology that is shared by at least half of the parent templates, at least two-thirds of the parent templates, at least three-fourths of the parent templates and in some embodiments almost all the parent templates. Furthermore, in some embodiments even a useful demarcation point is an area of homology that is shared by all parent templates.

In one embodiment, the gene reassembly process is exhaustively performed in order to generate a comprehensive library. In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the finished chimeric nucleic acid molecule set. At the same time, the order of assembly (ie the order of assembly of each building block in the 5 'or 3 sequence of each completed chimerized nucleic acid) in each combination is by design (or non-stochastic). Given the non-stochastic nature of this method, the possibility of unwanted by-products is greatly reduced.

In other embodiments, the method indicates that the gene reassembly process is performed systematically, for example to generate a systematically compartmentalized library, with compartments that can be systematically analyzed, such as one by one. In other words, through the selective and judicious use of specific nucleic acid building blocks coupled with the selective and judicious use of sequentially staggered assembly reactions, a design can be achieved where specific sets of progeny products are made in each of the various reaction vessels. This allows for a systematic examination and screening procedure. Therefore, it allows a large number of progeny molecules to be systematically examined in smaller groups.

Given its ability to perform chimerizations in a way that is highly flexible but comprehensive and also systematic, particularly when there is a low level of homology between the parent molecules, the disclosure provides for the generation of a library (or set) comprised of large numbers. of progeny molecules. Given the non-stochastic nature of the present gene reassembly invention, the progeny molecules generated in some embodiments comprise a library of finished chimerized nucleic acid molecules having a general assembly order which is chosen by design. In some embodiments, said generated library is comprised of more than 103 to more than 101,000 different progeny molecular species.

In some embodiments, a set of finished chimeric nucleic acid molecules produced as described is comprised of a polynucleotide encoding a polypeptide. In one embodiment, this polynucleotide is a gene, which can be a man-made gene. In another embodiment, this polynucleotide is a gene pathway, which may be a man-made gene pathway. In some embodiments, the disclosure indicates that one or more man-made genes generated by the invention can be incorporated into a man-made gene pathway, such as a pathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of this stage in which the building blocks are generated allows the design and introduction of nucleotides (such as one or more nucleotides, which may be eg codons or introns or regulatory sequences) that can be removed subsequently in an in vitro process (such as mutagenesis) or an in vivo process (such as using the gene splicing ability of a host organism). It is appreciated that in many cases the introduction of these nucleotides may also be desirable for many other reasons in addition to the possible benefit of creating a useful demarcation point.

Therefore, in some embodiments, a nucleic acid building block can be used to introduce an intron. Therefore, functional introns can be introduced into a man-made gene. In some embodiments, functional introns can be introduced into a man-made gene pathway. Accordingly, the disclosure provides for the generation of a chimeric polynucleotide that is a man-made gene that contains one (or more) artificially introduced introns.

Also disclosed is the generation of a chimeric polynucleotide which is a man-made gene pathway containing one (or more) artificially introduced introns. In some embodiments, artificially introduced introns are functional in one or more host cells to splice genes in the way that natural introns functionally serve in gene splicing. In addition, processes are described for producing polynucleotides containing man-made introns for introduction into host organisms for recombination and / or splicing.

A man-made gene produced as described herein can also serve as a substrate for recombination with another nucleic acid. Similarly, a man-made gene pathway produced as described herein can also serve as a substrate for recombination with another nucleic acid. In some embodiments, recombination is facilitated by or occurs in areas of homology between the intron-containing man-made gene and a nucleic acid, which serves as a recombination partner. In some embodiments, the recombination partner may also be a generated nucleic acid as disclosed herein, including a man-made gene or a man-made gene pathway. Recombination may be facilitated by or may occur in areas of homology that exist in one (or more) introns artificially inserted into the man-made gene.

In some embodiments, the synthetic gene reassembly method as disclosed herein uses a plurality of nucleic acid building blocks, each of which, in some embodiments, has two linkable ends. The two linkable ends in each nucleic acid building block may be two blunt ends (i.e. each having a zero nucleotide overhang) or in some embodiments a blunt end and a overhang or more, in some embodiments still two overhangs. In some embodiments, a protrusion useful for this purpose may be a 3 'overhang or a 5' overhang. Therefore, a nucleic acid building block can have a 3 'overhang or alternatively a 5' overhang or alternatively two 3 'overhangs or alternatively two 5' overhangs. The general order in which the nucleic acid building blocks assemble to form a finished chimeric nucleic acid molecule is determined by experimental design by design and is not random.

In some embodiments, a nucleic acid building block is generated by the chemical synthesis of two single-stranded nucleic acids (also called single-stranded oligos) and bringing them into contact to allow them to mate to form a single-stranded building block. double stranded nucleic acid. A double stranded nucleic acid building block can be of variable size. The sizes of these building blocks can be small or large. Exemplary sizes for the building block are in the range of 1 base pair (not including protrusions) to 100,000 base pairs (not including protrusions). Other exemplary size ranges are also provided, having lower limits from 1 bp to 10,000 bp (including each integer value in between) and upper limits from 2 bp to 100,000 bp (including each integer value in between).

There are many methods by which double stranded nucleic acid building blocks can be generated that are useful to the invention; and these are known in the art and can be easily performed by the skilled person. In some embodiments, a double-stranded nucleic acid building block is first generated by generating two single-stranded nucleic acids and allowing them to mate to form a double-stranded nucleic acid building block. The two strands of a double-stranded nucleic acid building block may be complementary at each nucleotide other than any that forms a overhang; therefore they contain no mismatches, apart from any overhang. In another embodiment, the two strands of a double stranded nucleic acid building block are complementary in fewer nucleotides apart from any one that forms a protrusion. Therefore, in accordance with this embodiment, a double-stranded nucleic acid building block can be used to introduce codon degeneracy. In some embodiments, codon degeneracy is introduced using the site saturation mutagenesis described herein, using one or more N, N, G / T cassettes, or alternatively using one or more N, N, N cassettes.

The in vivo recombination method disclosed herein can be performed blindly in a pool of unknown hybrids or alleles of a specific polynucleotide or sequence. However, it is not necessary to know the DNA or RNA sequence of the specific polynucleotide. The approach of using recombination within a mixed population of genes can be useful for the generation of any useful protein, for example, an aldolase according to the invention or a variant thereof. This approach can be used to generate proteins that have specificity or altered activity. This approach may also be useful for the generation of hybrid nucleic acid sequences, eg, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes. Therefore this approach can be used to generate genes that have increased rates of expression. This approach may also be useful in the study of repetitive DNA sequences. Finally, this approach may be useful for making ribozymes or aptamers disclosed herein.

Some embodiments disclosed herein are directed to the use of repeated cycles of reductive rearrangement, recombination, and selection that allow for directed molecular evolution of highly complex linear sequences, such as DNA, RNA, or protein through recombination.

Optimized directed evolution system

There is disclosed a non-stochastic gene modification system called "optimized targeted evolution system" to generate polypeptides, such as aldolase enzymes or antibodies, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, with novel properties or altered. In some embodiments, optimized targeted evolution is directed to the use of repeated cycles of reductive rearrangement, recombination, and selection that allow for targeted molecular evolution of nucleic acids through recombination.

Optimized Targeted Evolution allows the generation of a large population of evolved chimeric sequences, where the generated population is significantly enriched for sequences that have a predetermined number of crossover events. A crossover event is a point in a chimeric sequence where a change in sequence occurs from one parent variant to another parent variant. Said point is normally at the junction where the oligonucleotides of two parents bind to form a single sequence. This method allows the calculation of correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides greater control over the choice of chimeric variants that have a predetermined number of crossover events.

Furthermore, this method provides convenient means to explore a large amount of the protein variant space possible compared to other systems. Previously, if for example 1013 chimeric molecules were generated during a reaction, it would be extremely difficult to analyze such a high number of chimeric variants for a particular activity. Furthermore, a significant portion of the progeny population would have a very high number of crossover events resulting in proteins that were less likely to have increased levels of a particular activity. Using these methods, the population of chimeric molecules can be enriched for these variants that have a particular number of crossover events. Therefore, while 1,013 chimeric molecules can still be generated during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events. Since the resulting progeny population can be biased to have a predetermined number of crossover events, the limits on functional variety between chimeric molecules are reduced. This provides a more manageable number of variables when calculating which oligonucleotide of the original parental polynucleotides may be responsible for affecting a particular trait.

One method of creating a chimeric progeny polynucleotide sequence is to create oligonucleotides that correspond to fragments or portions of each parent sequence. Each oligonucleotide in some embodiments includes a unique overlapping region so that mixing the oligonucleotides results in a new variant that has each oligonucleotide fragment assembled in the correct order. Alternative protocols for carrying out these methods in accordance with the invention can be found in US Patent No. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.

The number of oligonucleotides generated for each parental variant has a relationship to the total number of resulting crosses in the chimeric molecule that is created at the end. For example, three parental nucleotide sequence variants may be provided to undergo a ligation reaction so as to find a chimeric variant having eg higher activity at high temperature. As an example, a set of 50 oligonucleotide sequences can be generated corresponding to each portion of each parental variant. Consequently, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each generated chimeric polynucleotide contains oligonucleotides of each parental variant in alternate order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar amount, it is likely that at some positions the oligonucleotides of the same parent polynucleotide will ligate close to each other and therefore not result in a crossover event. If the concentration of each oligonucleotide from each parent is held constant during any ligation step in this example, there is a possibility of Y (assuming 3 parents) that an oligonucleotide of the same parental variant binds within the chimeric sequence and does not produce a crossing.

Therefore, a probability density function (PDF) can be determined to predict the population of crossover events that are likely to occur during each stage in a ligation reaction given a fixed number of parental variants, a number of oligonucleotides corresponding to each variant and the concentrations of each variant during each stage in the ligation reaction. The statistics and math behind determining the PDF are outlined below. Using these methods, such a probability density function can be calculated, and thus enriching the chimeric progeny population for a predetermined number of crossing events resulting from a particular ligation reaction. Furthermore, a target number of crossing events can be predetermined and the system can then be programmed to calculate the starting amounts of each parent oligonucleotide during each stage in the ligation reaction to result in a probability density function that focuses on the number default crossover event. These methods are directed to the use of repeated cycles of reductive rearrangement, recombination, and selection that allow for directed molecular evolution of a nucleic acid by encoding a polypeptide through recombination. The system allows the generation of a large population of evolved chimeric sequences, where the generated population is significantly enriched for sequences that have a predetermined number of crossover events. A crossover event is a point in a chimeric sequence where a change in sequence occurs from one parent variant to another parent variant. Said point is normally at the junction where the oligonucleotides of two parents bind to form a single sequence. This method allows the calculation of correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides greater control over the choice of chimeric variants that have a predetermined number of crossover events.

Furthermore, these methods provide a convenient means of exploring a large amount of the protein variant space possible compared to other systems. Using the methods described herein, the population of chimeric molecules can be enriched for these variants that have a particular number of crossover events. Therefore, while 1,013 chimeric molecules can still be generated during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events. Since the resulting progeny population can be biased to have a predetermined number of crossover events, the limits on functional variety between chimeric molecules are reduced. This provides a more manageable number of variables when calculating which oligonucleotide of the original parental polynucleotides may be responsible for affecting a particular trait.

In some embodiments, the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides that correspond to fragments or portions of each parent sequence. Each oligonucleotide in some embodiments includes a unique overlapping region so that mixing the oligonucleotides results in a new variant that has each oligonucleotide fragment assembled in the correct order. See United States Patent No. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.

Determination of crossover events

Embodiments include a system and software that receive a desired crossover probability density (PDF) function, the number of parent genes to reassemble, and the number of fragments in the reassembly as inputs. The output of this program is a "PDF fragment" that can be used to determine a recipe to produce reassembled genes and the estimated crossover PDF of those genes. The processing described herein is performed in some embodiments in MATLAB ™ (The Mathworks, Natick, Mass.) A programming language and development environment for technical computing.

Iterative processes

Any process disclosed herein can be repeated, such as a nucleic acid encoding a new or altered phenotype of an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, can be identified, isolated, modified again, reanalyze for activity. This process can be repeated until a desired phenotype is designed. For example, an entire biochemical anabolic or catabolic pathway can be designed in a cell, including, for example, an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase.

Similarly, if it is determined that a particular oligonucleotide in no way affects the desired trait (such as a new aldolase enzyme phenotype, such as pyruvate aldolase, such as HMG and / or KHG aldolase), it can be removed as a variable by synthesizing oligonucleotides. larger parentals that include the sequence to be removed. Since incorporating the sequence into a larger sequence avoids any crossover events, there will be no further variation of this sequence on the progeny polynucleotides. This iterative practice of determining which oligonucleotides are most related to the desired trait, and which are not related, allows for a more efficient exploration of all possible protein variants that may provide a particular trait or activity.

Transposition in vivo

In various embodiments, the in vivo transposition of molecules is used in methods disclosed herein to provide variants of the disclosed polypeptides, such as antibodies disclosed herein or aldolase enzymes disclosed herein, such as pyruvate aldolase, HMG and / or KHG aldolase and the like. Transposition in vivo can be performed using the natural property of cells to recombine multimers. While in vivo recombination provided the primary natural route for molecular diversity, genetic recombination remains a relatively complex process involving 1) recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasm; and finally 3) the resolution of chiasm in discrete recombined molecules. Chiasm formation requires recognition of homologous sequences.

Also described is a method of producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide. In some embodiments, the method can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide (such as one, or both, being an aldolase enzyme coding sequence, such as pyruvate aldolase, such as HMG and / or KHG aldolase according to the invention) that share at least one region of partial sequence homology in a suitable host cell. The partial sequence homology regions promote processes that result in sequence rearrangement producing a hybrid polynucleotide. The term "hybrid polynucleotide" as used herein is any nucleotide sequence that results from the method and contains sequence of at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events that promote sequence integration between DNA molecules. Furthermore, such hybrid polynucleotides can result from intramolecular reductive rearrangement processes that use repeated sequences to alter a nucleotide sequence within a DNA molecule.

In some embodiments, live rearrangement focuses on "intermolecular" processes collectively called "recombination"; which in bacteria is generally seen as a “RecA-dependent” phenomenon. In some embodiments, the method may rely on recombination processes of a host cell to recombine and rearrange sequences, or the ability of cells to mediate reducing processes to decrease the complexity of near-repeat sequences in the cell by deletion. This “reductive rearrangement” process occurs through an independent “intramolecular” RecA process.

In other embodiments, new polynucleotides can be generated by the reductive rearrangement process. The method involves generating constructs containing consecutive sequences (original coding sequences), inserting them into an appropriate vector, and subsequently introducing them into an appropriate host cell. The rearrangement of individual molecular identities occurs by combinatorial processes between consecutive sequences in the construct having regions of homology or between nearly repeated units. The rearrangement process recombines and / or reduces the extension complexity of the repeated sequences and results in the production of new molecular species. Various treatments can be applied to improve the rearrangement rate. These could include treatment with ultraviolet light, or chemicals that damage DNA and / or the use of host cell lines that show improved levels of "genetic instability." Therefore the rearrangement process may involve homologous recombination or the natural property of near-repeat sequences to direct its own evolution.

Repeated or "near repeat" sequences have a role in genetic instability. In some embodiments, "near repeats" are repeats that are not limited to their original unit structure. Nearly repeating units can be presented as an array of sequences in a construct; consecutive units of similar sequences. Once linked, the junctions between consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level. The elimination process that the cell performs to reduce the complexity of the resulting construct operates between the nearly repeated sequences. Nearly repeating units provide a virtually limitless repertoire of templates upon which slip events can occur. In some embodiments, the constructs containing the quasi-repeats therefore effectively provide sufficient molecular elasticity such that deletion (and potentially insertion) events can occur virtually anywhere within quasi-repeating units.

When quasi-repeated sequences are all ligated in the same orientation, for example head-to-tail or vice versa, the cell cannot distinguish individual units. Therefore, the reducing process can occur in all sequences. In contrast, when for example units are presented head-to-head, rather than head-to-tail, the inversion delineates the endpoint of the adjacent unit so that the formation of elimination will favor the loss of discrete units. Therefore, it is preferred with the present method that the sequences be in the same orientation. Random orientation of quasi-repeated sequences will result in loss of rearrangement efficiency, while consistent sequence orientation will offer the highest efficiency. However, while having fewer contiguous sequences in the same orientation reduces efficiency, it can still provide enough elasticity for effective recovery of new molecules. Constructs can be performed with the sequences nearly repeated in the same orientation to allow for greater efficiency.

Sequences can be assembled in a head-to-tail orientation using any of a variety of methods, including the following:

•

a) Primers including a poly-A head and poly-T tail can be used which when made single stranded would provide orientation. This is accomplished by having the first new primer bases made from RNA and therefore easily removed RNAseH.

•

b) Primers including single restriction cleavage sites can be used. Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required.

•

c) The few internal bases of the primer could be thiolated and an exonuclease could be used to produce properly tailed molecules.

In some embodiments, the recovery of the rearranged sequences depends on the identification of cloning vectors with a reduced repetitive index (RI). The rearranged coding sequences can then be recovered by amplification. Products recline and express themselves. Recovery of cloning vectors with reduced IR may be affected by:

1) The use of vectors only stably maintained when the construct is reduced in complexity

2) The physical recovery of shortened vectors using physical procedures. In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on agarose gel or column with a low molecular weight cutoff using standard procedures.

3) Recovery of vectors containing interrupted genes that can be selected when the insert size is reduced.

4) The use of direct selection techniques with an expression vector and the appropriate selection.

The coding sequences (eg genes) of related organisms can demonstrate a high degree of homology and encode quite diverse protein products. These types of sequences are particularly useful as near repeats. However, while the examples illustrated below demonstrate the rearrangement of nearly identical (near repeats) original coding sequences, this process is not limited to such nearly identical repeats.

The following example demonstrates an exemplary method according to the invention. Coding nucleic acid sequences (near repeats) derived from three (3) unique species are described. Each sequence encodes a protein with a different set of properties. Each of the sequences differs by a single or a few base pairs at a single position in the sequence. The quasi-repeated sequences are amplified separately or together and ligated into random assemblies such that possible permutations and combinations are available in the population of linked molecules. The number of quasi-repeating units can be controlled by assembly conditions. The average number of quasi-repeating units in a construct is defined as the repeating index (RI).

Once formed, the constructs may or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell. The cells are then propagated and a "reductive rearrangement" is performed. The rate of the reductive rearrangement process can be stimulated by introducing DNA damage if desired. It is not relevant whether the reduction in IR is mediated by elimination formation between repeating sequences by an "intramolecular" mechanism, or mediated by events similar to recombination through "intermolecular" mechanisms. The end result is a rearrangement of the molecules in all possible combinations.

Optionally, the method comprises the additional step of analyzing the library members of the transposed pool to identify individual rearranged library members that have the ability to bind or otherwise interact, or catalyze a particular reaction (such as a catalytic domain of a enzyme) with a predetermined macromolecule, such as for example a protein receptor, an oligosaccharide, virion or other predetermined compound or structure.

The polypeptides that are identified from such libraries can be used for therapeutic, diagnostic, research, and related purposes (such as catalysts, solutes to increase the osmolarity of an aqueous solution, and the like) and / or can undergo one or more additional cycles of transposition and / or selection.

In other embodiments, it is envisioned that before or during recombination or rearrangement, polynucleotides generated by the method disclosed herein will be subjected to agents or processes that promote the introduction of mutations into the original polynucleotides. The introduction of such mutations would increase the diversity of the hybrid polynucleotides and polypeptides encoded from there. Agents or processes that promote mutagenesis may include but are not limited to: (+) - CC-1065, or a synthetic analog such as (+) - CC-1065- (N3-Adenine (see Sun and Hurley, (1992 ); an N-acetylated or deacetylated 4'-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis (see, for example, van de Poll et al. (1992)); or an N-acetylated 4-aminobiphenyl adduct or deacetylated capable of inhibiting DNA synthesis (see also, van de Poll et al. (1992), pp. 751-758); trivalent chromium, a trivalent chromium salt, an adduct of aromatic polycyclic hydrocarbon (PAH) DNA capable of inhibiting DNA replication, such as 7-bromomethyl-benz [a] anthracene ("BMA"), tris (2,3-dibromopropyl) phosphate ("Tris-BP"), 1,2-dibromo-3-chloropropane ( "DBCP"), 2-bromoacrolein (2BA), benzo [a] pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a platinum (II) halogen salt, N-hydroxy-2 -amino-3-methylimidazo [4,5-f] -quinoline ("N-hydroxy-IQ") and N-hydroxy-2-amino-1-methyl6-phenylimidazo [4,5-f] -pyridine ("N -hydroxy- PhIP ”). Examples of means to slow down or stop PCR amplification consist of UV light (+) - CC-1065 and (+) - CC-1065- (N-3-Adenine). Means encompassed in particular are DNA or polynucleotide adducts comprising the DNA adducts of the polynucleotides or the polynucleotide pool, which may be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing.

Also disclosed is a method of producing recombinant proteins that have biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions that provide for the production of hybrid or rearranged polynucleotides.

Production of sequence variants

Additional methods for making sequence variants of nucleic acid sequences (such as an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase) disclosed herein are also disclosed. In some embodiments, the invention also provides additional methods for isolating aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase using the nucleic acids and polypeptides disclosed herein. Variants of an aldolase are described, such as pyruvate aldolase, HMG and / or KHG aldolase enzyme coding sequence (such as a gene, cDNA or message) disclosed herein, which can be altered by any means, including for example random methods or stochastic or non-stochastic or "directed evolution" methods as described above.

Isolated variants can be natural. The variant can also be created in vitro. Variants can be created using genetic manipulation techniques such as site-directed mutagenesis, random chemical mutagenesis, exonuclease III removal procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures. Other methods of making variants are also known to those of skill in the art. This includes procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids encoding polypeptides that have characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences are generated and characterized that have one or more nucleotide differences from the sequence obtained from the natural isolate. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids of the natural isolates.

For example, variants can be created using error prone PCR. In some error-prone embodiments of PCR, PCR is performed under conditions where the copy fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained over the entire length of the PCR product. Error prone PCR is described as in Leung, D. W. et al., (1989) Technique 1: 11-15; and Caldwell, R.C. & Joyce, G.F., (1992) PCR Methods Applic. 2: 28-33. Briefly, in such procedures, the nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase, and an appropriate concentration of dNTP to achieve a high point mutation rate over the entire length. of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmoles from each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% Gelatin, 7mM MgCl2, 0.5mM MnCl2, 5 Taq Polymerase Units, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP and 1mM dTTP. PCR can be performed for 30 cycles of 94 ° C for 1 minute, 45 ° C for 1 minute, and 72 ° C for 1 minute. However, it will be appreciated that these parameters may vary as appropriate. The mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.

In some embodiments, variants are created using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described for example in Reidhaar-Olson (1988) Science 241: 53-57. Briefly, in such procedures a plurality of double-stranded oligonucleotides having one or more mutations to be introduced into the cloned DNA is synthesized and inserted into the cloned DNA to be mutagenized. In some embodiments, clones containing the mutagenized DNA are recovered, expressed, and the activities of the polypeptide encoded therein are evaluated.

Another method of generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in the art, such as in US Patent 5,965,408.

In some embodiments, sexual PCR mutagenesis is an example of a method of generating variants disclosed herein. In some embodiments of sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of a different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation. crossing by primer extension in a PCR reaction. Sexual PCR mutagenesis is described for example in Stemmer (1994) Proc. Natl. Acad. Sci. United States 91: 10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with Dnase to generate fragments having an average size of 50-200 nucleotides. Fragments of the desired average size are purified and resuspended in a PCR mixture. PCR is performed under conditions that facilitate recombination between nucleic acid fragments. For example, PCR can be performed by resuspending the purified fragments at a concentration of 10-30 ng / µl in a 0.2mM solution of each dNTP, 2.2mM MgCl2, 50mM KCL, 10mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5 units of Taq polymerase per 100 µl of reaction mixture are added and PCR is performed using the following regimen: 94 ° C for 60 seconds, 94 ° C for 30 seconds, 50-55 ° C for 30 seconds, 72 ° C for 30 seconds (30-45 times) and 72 ° C for 5 minutes. However, it will be appreciated that these parameters may vary as appropriate. In some embodiments, the oligonucleotides can be included in the PCR reactions. In other embodiments, the Klenow fragment of DNA polymerase I can be used in a first set of PCR reactions and Taq polymerase can be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the encoding polypeptides are evaluated.

In some embodiments, the variants are created by in vivo mutagenesis. In some embodiments, random mutations in a sequence of interest are generated by propagating the sequence of interest into a bacterial strain, such as the E. coli strain that carries mutations in one or more of the DNA repair pathways. Such "mutant" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Mutant strains suitable for use for in vivo mutagenesis are described in PCT Publication No. WO 91/16427, published Oct. 31. 1991 entitled "Methods for Phenotype Creation from Multiple Gene Populations".

Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double-stranded DNA molecule is replaced by a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains a fully and / or partially randomized native sequence.

Recursive assembly mutagenesis can also be used to generate variants. Recursive assembly mutagenesis is an algorithm for protein manipulation (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive assembly mutagenesis is described for example in Arkin (1992) Proc. Natl. Acad. Sci. United States 89: 7811-7815.

In some embodiments, the variants are created using exponential assembly mutagenesis. Exponential assembly mutagenesis is a process to generate combinatorial libraries with a high percentage of unique and functional mutants, where small groups of residues are randomized in parallel to identify, at each altered position, amino acids that lead to functional proteins. Exponential assembly mutagenesis is described for example in Delegrave (1993) Biotechnology Res. 11: 1548-1552. Random and site-directed mutagenesis is described, for example, in Arnold (1993) Current Opinion in Biotechnology 4: 450-455.

In some embodiments, variants are created using rearrangement procedures where portions of a plurality of nucleic acids encoding different polypeptides are fused to create chimeric nucleic acid sequences encoding chimeric polypeptides as described in US Patent 5,965,408. , presented on Jul 9. 1996, entitled "DNA Reassembly Method by Disruption of Synthesis" and US Patent No. 5,939,250, filed May 22, 1996, entitled "Production of Enzymes Having Desired Activities by Mutagenesis".

Variants of the polypeptides disclosed herein may be variants in which one or more of the amino acid residues of the polypeptides of the sequences disclosed herein are replaced with a conserved or non-conserved amino acid residue (in some embodiments, a conserved amino acid residue) and said substituted amino acid residue may or may not be encoded by the genetic code.

In some embodiments, conservative substitutions are those that substitute a given amino acid in a polypeptide for another amino acid of similar characteristics. In some embodiments, the conservative substitutions disclosed herein comprise the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine, and Isoleucine with another aliphatic amino acid; replacing a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue having an amide group, such as Asparagine and Glutamine, with another residue having an amide group; exchanging a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residues of a polypeptide disclosed herein include a substituent group. In some embodiments, other variants are those in which the polypeptide associates with another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol). Additional variants are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence, or a sequence that facilitates purification, enrichment, or stabilization of the polypeptide.

In some embodiments, the fragments, derivatives, and analogs retain the same biological function or activity as the polypeptides disclosed herein and sequences substantially identical thereto. In other embodiments, the fragment, derivative, or analog includes a proprotein, such that the fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide.

Codon optimization to achieve high levels of protein expression in host cells

Methods are described for modifying nucleic acids encoding an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, to modify (such as optimize) the use of codons. Methods for modifying codons in a nucleic acid encoding an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase to increase or decrease its expression in a host cell are further described. Nucleic acids encoding an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, are modified to increase their expression in a host cell, enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, thus modified , and methods for preparing the aldolase enzymes, such as pyruvate aldolase, such as HMG and / or modified KHG aldolase. The method comprises identifying a "non-preferred" or "least preferred" codon in nucleic acid encoding aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase, and replacing one or more of these non-preferred or less preferred codons with a "preferred codon" encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid. A preferred codon is an underrepresented codon in gene encoding sequences in the host cell and a non-preferred or less preferred codon is an underrepresented codon in gene encoding sequences in the host cell.

Host cells for expressing the nucleic acids, expression cassettes, and vectors disclosed herein include bacteria, yeast, fungi, plant cells, insect cells, and mammalian cells (see discussion above). Therefore, there are methods to optimize codon usage in all of these cells, codon-altered nucleic acids, and polypeptides made by codon-altered nucleic acids. Examples of host cells include gram negative bacteria such as Escherichia coli; Gram positive bacteria, such as Streptomyces sp., Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis, Bacillus cereus. Examples of host cells also include eukaryotic organisms, such as various yeasts, such as Saccharomyces sp., Including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines. insect cell phones. Therefore, nucleic acids and polypeptides optimized for expression in these organisms and species are also described.

For example, codons of a nucleic acid encoding an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell other than those bacteria from which the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase are derived, a yeast, a fungus, a plant cell, an insect cell or a mammalian cell. Methods for optimizing codons are well known in the art, see US Patent No. 5,795,737; Baca (2000) Int. J. Parasitol. 30: 113-118; Hale (1998) Protein Expr. Purif 12: 185-188; Narum (2001) Infect. Immun. 69: 7250-7253. Also see Narum (2001) Infect. Immun. 69: 7250-7253, which describes optimization codons in mouse systems; Outchkourov (2002) Protein Expr. Purif. 24: 18-24, which describes optimization codons in yeast; Feng (2000) Biochemistry 39: 15399-15409, which describes optimization codons in E. coli; Humphreys (2000) Protein Expr. Purif. 20: 252-264, which describes the use of optimization codons that affects secretion in E. coli.

Transgenic non-human animals

Transgenic non-human animals comprising a nucleic acid, a polypeptide (such as an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase), a cassette or expression vector or a transfected or transformed cell disclosed in the invention are also disclosed. Present. Methods for making and using these transgenic non-human animals are also disclosed.

Transgenic non-human animals can be, for example, dogs, goats, rabbits, sheep, horses, fish, pigs (including all pigs, pigs, and related animals), cows, rats, and mice, which comprise the nucleic acids disclosed in the Present. These animals can be used for example in in vivo models to study the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, the activity or as models to analyze agents that change the enzyme aldolase, such as pyruvate aldolase, such like HMG and / or KHG aldolase activity in vivo. Coding sequences for polypeptides to be expressed in transgenic non-human animals can be designed to be constitutive or under the control of tissue-specific, developmental-specific, or inducible transcriptional regulatory factors.

Transgenic non-human animals can be designed and generated using any method known in the art; see United States Patents No. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571, which describe the making and use of transgenic cells and eggs and transgenic mice, rats, rabbits, sheep, pigs, chickens, goats, fish, and cows. See also for example Pollock (1999) J. Immunol. Methods 231: 147-157, which describes the production of recombinant proteins in the milk of transgenic dairy-producing animals; Baguisi (1999) Nat. Biotechnol. 17: 456-461, which demonstrates the production of transgenic goats. United States Patent No. 6,211,428, describes the making and use of transgenic non-human mammals expressing in their brains a nucleic acid construct comprising a DNA sequence. US Patent 5,387,742, describes the injection of recombinant or synthetic cloned DNA sequences into fertilized mouse eggs, the implantation of eggs injected into pseudo-pregnant females, and the term growth growth of transgenic mice. United States Patent No. 6,187,992 describes the making and use of a transgenic mouse.

"Knockout animals" can also be used to perform the methods disclosed herein. For example, in some embodiments, the transgenic or modified animals disclosed herein comprise a "knockout animal", such as a "knockout mouse", manipulated not to express an endogenous gene, which is replaced by a gene that expresses an aldolase enzyme , such as pyruvate aldolase, HMG and / or KHG aldolase disclosed herein, or a fusion protein comprising an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase disclosed herein

Transgenic plants and seeds

The invention provides transgenic plants and seeds comprising a nucleic acid, a polypeptide (such as an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase), a cassette or expression vector, or a transfected or transformed cell. The invention also provides plant products or derivatives, such as fruits, oils, seeds, leaves, extracts, and the like, including any plant part, comprising a nucleic acid and / or polypeptide (such as a xylanase) disclosed herein, such like where the nucleic acid

or polypeptide disclosed herein is heterologous to the plant, plant part, seed, etc. The transgenic plant (which includes plant parts, fruits, seeds, etc.) can be dicotyledonous or monocotyledonous. Methods for making and using these transgenic plants and seeds are also disclosed. The transgenic plant or plant cell expressing a polypeptide disclosed herein can be constructed according to any method known in the art. See, for example, United States Patent No. 6,309,872.

The nucleic acids and expression constructs disclosed herein can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or nucleic acids or expression constructs can be episomes. The introduction into the genome of a desired plant may be such that the production of enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase from the host is regulated by endogenous transcriptional or translational control elements. Also described are "knockout plants" where insertion of the gene sequence by for example homologous recombination has altered the expression of the endogenous gene. Means for generating "knockout" plants are well known in the art, see (1998) Proc Natl. Acad. Sci. United States 95: 4368-4373; Miao (1995) Plant J 7: 359-365. See the discussion on transgenic plants below.

The nucleic acids disclosed herein can be used to confer desired traits on essentially any plant, such as starch-producing plants, such as potato, tomato, soybean, beet, corn, wheat, rice, barley, and the like. The nucleic acids disclosed herein can be used to manipulate metabolic pathways of a plant to optimize or alter expression of the enzyme aldolase host, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The nucleic acids disclosed herein can change the levels of expression or activity or alter the characteristics of naturally occurring compounds or enzymes in a plant. Alternatively, an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, disclosed herein can be used in the production of a transgenic plant to produce a compound not naturally produced by said plant. This can reduce production costs or create a new product.

In some embodiments, the first step in the production of a transgenic plant involves making an expression construct for expression in a plant cell. These techniques are well known in the art. They may include selecting and cloning a promoter, a coding sequence to facilitate efficient ribosome binding to mRNA, and selecting the appropriate gene terminator sequences. An example of a constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to signals in the internal or external environment of the plant. An example of a light inducible promoter is the cab gene promoter, which encodes the major chlorophyll a / b binding protein.

In some embodiments, the nucleic acid is modified to achieve increased expression in a plant cell. For example, a sequence disclosed herein is likely to have a higher percentage of A-T nucleotide pairs compared to that seen in the plant, some of which prefer G- nucleotide pairs.

C. Therefore, A-T nucleotides in the coding sequence can be replaced with G-C nucleotides without significantly changing the amino acid sequence to improve production of the gene product in plant cells.

The selectable marker gene can be added to the gene construct to identify plant cells or tissues that have successfully integrated the transgene. This may be necessary because achieving gene incorporation and expression in plant cells is a rare event, occurring only in a low percentage of targeted tissues or cells. The selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown in a medium containing the appropriate antibiotic or herbicide. For other inserted genes, marker genes also require promoter and termination sequences for proper operation.

In some embodiments, making transgenic plants or seeds comprises incorporating sequences disclosed herein and optionally marker genes into a target expression construct (such as a plasmid) along with positioning of the promoter and terminator sequences. This may involve transferring the modified gene into the plant through a suitable method. For example, a construct can be directly introduced into plant cell genomic DNA using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be directly introduced into plant tissue using ballistic methods, such as bombardment of DNA particles. For example, see Christou (1997) Plant Mol. Biol. 35: 197-203; Pawlowski (1996) Mol. Biotechnol. 6: 17-30; Klein (1987) Nature 327: 70-73; Takumi (1997) Genes Genet. Syst. 72: 63-69, which describes the use of particle bombardment to introduce transgenes into wheat; and Adam (1997) supra, for the use of particle bombardment to introduce YAC into plant cells. For example, Rinehart (1997) supra, used particle bombardment to generate transgenic cotton plants. Particle acceleration apparatus are described in US Patent No. 5,015,580 and the available Bio-Rad (Biolistics) PDS-2000 particle acceleration instrument (Bio-Rad, Hercules, CA); see also John, US Patent No. 5,608,148 and Ellis, US Patent No. 5,681,730, which describes the transformation of gymnosperms measured by particles.

In some embodiments, the protoplasts can be immobilized and injected with nucleic acids, such as an expression construct. Although plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast-derived calli. Organized tissues can be transformed with naked DNA using the gene gun technique, where the DNA is coated in tungsten microprojectiles, fired 1/100 the size of cells that carry DNA deep into cells and organelles. The transformed tissue is then induced to usually regenerate by somatic embryogenesis. This technique was successful in several cereal species including corn and rice.

Nucleic acids such as expression constructs can also be introduced into plant cells using recombinant viruses. Plant cells can be transformed using viral vectors, such as vectors derived from tobacco mosaic virus (Rouwendal (1997) Plant Mol. Biol. 33: 989-999), see Porta (1996) "Use of viral replicons for the expression of genes in plants ”, Mol. Biotechnol. 5: 209-221.

Alternatively, nucleic acids, such as an expression construct, can be combined with suitable DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will target insertion of the construct and adjacent marker into the DNA of the plant cell when the cell is infected by bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarmament and the use of binary vectors, are well described in the scientific literature. See Horsch (1984) Science 233: 496-498; Fraley (1983) Proc. Natl. Acad. Sci. United States 80: 4803 (1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995). DNA in an A. tumefaciens cell is contained on the bacterial chromosome, as well as in another structure known as a Ti (tumor inducing) plasmid. The Ti plasmid contains a DNA elongation called T-DNA (˜20 kb in length) that is transferred to the plant cell in the infection process and a series of vir (virulence) genes that direct the infection process. A. tumefaciens can only infect a plant through wounds: when a root or plant stem is wounded it gives certain chemical signals in response to which the A. tumefaciens vir genes become activated and direct a series of events necessary for transfer of the T-DNA from the Ti plasmid to the plant chromosome. The T-DNA then enters the plant cell through the wound. One speculation is that the T-DNA waits until the plant DNA is replicated or transcribed, then inserted into the exposed plant DNA. In order to use A. tumefaciens as a transgene vector, the T-DNA tumor-inducing section has to be removed while retaining the T-DNA border regions and vir genes. The transgene is then inserted between the T-DNA border regions, where it is transferred to the plant cell and integrated into the plant chromosomes.

The disclosure provides for the transformation of monocot plants using the nucleic acids disclosed herein, including important cereals, see Hiei (1997) Plant Mol. Biol. 35: 205-218. See also, Horsch, Science (1984) 233: 496; Fraley (1983) Proc. Natl. Acad. Sci. United States 80: 4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32: 1135-1148, which describe the integration of T-DNA into genomic DNA. See also D'Halluin, US Patent No. 5,712,135, which describes a process for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal, or other monocotyledonous plant.

In some embodiments, the third stage involves the selection and regeneration of whole plants capable of transmitting the embedded target gene to the next generation. Such regeneration techniques can use the manipulation of certain phytohormones in a tissue culture growth medium. In some embodiments, the method uses a biocide and / or herbicide marker that was introduced along with the desired nucleotide sequences. Plant regeneration of cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant corns, explants, organs, or parts thereof. Such regeneration techniques are generally described in Klee (1987) Ann. Rev. of Plant Phys. 38: 467-486. To obtain whole plants of transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are spawned and produce seeds, progeny evaluation begins.

In some embodiments, after the expression cassette is stably incorporated into transgenic plants, it can be introduced into other plants by sexual crossing. Any of a number of breeding techniques can be used, depending on the species to be crossed. Since the transgenic expression of the nucleic acids disclosed herein leads to phenotypic changes, plants comprising the nucleic acids disclosed herein can be sexually crossed with a second plant to obtain a final product. Therefore, the seed disclosed herein may be derived from a cross between two transgenic plants disclosed herein, or a cross between a plant disclosed herein and another plant. The desired effects (such as the expression of the polypeptides disclosed herein to produce a plant in which flowering behavior is altered) can be enhanced when both parent plants express the polypeptides (such as an enzyme aldolase, pyruvate aldolase, HMG and / or HHG aldolase) disclosed herein. The desired effects can be passed on to future plant generations by standard propagation means.

In some embodiments, the nucleic acids and polypeptides disclosed herein are expressed or inserted into any plant or seed. The transgenic plants disclosed herein can be dicotyledonous or monocotyledonous. Examples of monocotyledonous transgenic plants disclosed herein according to the invention are grasses, such as prairie grass (blue grass, Poa), forage grass such as fescue, lolium, temperate grass, such as Agrostis, and cereals, such as wheat , oats, rye, barley, rice, sorghum and corn. Examples of transgenic dicotyledonous plants disclosed herein are tobacco, legumes, such as lupines, potatoes, sugar beets, peas, beans, and soybeans, and cruciferous plants (family Brassicaceae) such as cauliflower, rapeseed, and the related related model organism Arabidopsis thaliana. Therefore, the transgenic plants and seeds disclosed herein include a wide range of plants, including but not limited to species of the genus Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaceis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Loluim, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotza, Ogo Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanumm, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna and Zea.

In alternative embodiments, the nucleic acids disclosed herein are expressed in plants containing fiber cells, including, for example, cotton, silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosata bush, krascheninnikovia , raft, ramie, kenaf, hemp, Jamaican rose, jute, sisal, abaca and flax. In alternative embodiments, the transgenic plants disclosed herein can be members of the genus Gossypium, including members of any Gossypium species, such as G. arboreum; G. herbaceum, G. barbadense and G. hirsutum.

Transgenic plants to be used to produce large amounts of the polypeptides (such as an aldolase enzyme or antibody, pyruvate aldolase, HMG and / or KHG aldolase) disclosed herein are described. For example, see Palmgren (1997) Trends Genet. 13: 348; Chong (1997) Transgenic Res. 6: 289-296 (production of human milk protein beta-casein in transgenic potato plants using a auxin-inducible bidirectional manopine synthase promoter (mas1 ', 2') with methods of transformation of leaf disc mediated by Agrobacterium tumefaciens).

Using known procedures, an expert can analyze plants disclosed herein by detecting the increase or decrease of RNA, transgene, or protein in transgenic plants. Means for detecting and quantifying mRNA, or protein are well known in the art.

Polypeptides and peptides

Isolated, synthetic or recombinant polypeptides are disclosed that have a sequence identity (such as at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76% , 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more or a sequence identity or full homology (100%)) with respect to a sequence disclosed herein, such as proteins having a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 , SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO : 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 , SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO : 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162 , SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO : 196, SEQ ID NO: 1 98, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 and enzymatically active fragments thereof. The percentage sequence identity can be in the total length of the polypeptide or the identity can be in a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 , 500, 550, 600, 650, 700 or more waste.

Polypeptides according to some embodiments disclosed herein may also be shorter than the full length of the polypeptides. Polypeptides (peptides, fragments) can range from about 5 to the full length of a polypeptide, such as an enzyme, such as an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase; exemplary sizes being about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues, such as contiguous residues of an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed in Present. The peptides disclosed herein (such as a subsequence of a polypeptide disclosed herein) may be useful as, for example, labeling probes, active sites of antigens (immunogens), tolerogens, motifs, aldolase, such as pyruvate aldolase enzyme , such as HMG and / or KHG aldolase (such as "catalytic domains"), signal sequences and / or prepro domains.

In other embodiments, the polypeptides disclosed herein that have aldolase activity, such as pyruvate aldolase activity, such as HMG and / or KHG aldolase are members of a genus of polypeptides that share specific structural elements, such as amino acid residues, which correlate with aldolase activity, including pyruvate activity such as, but not limited to, HMG and / or KHG aldolase activity. These shared structured elements can be used for routine generation of aldolase, such as pyruvate aldolase variants, such as HMG and / or KHG aldolase. These shared structural elements of aldolase, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase, disclosed herein, can be used as a guide for routine generation of aldolase, such as pyruvate aldolase variants, such as HMG and / or KHG aldolase within the scope of the polypeptide genus disclosed herein.

As used herein, the term "aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase" encompasses any polypeptide or enzymes capable of catalyzing the aldol addition reaction or the retro-aldol reaction (such as polypeptides disclosed herein, see also Table 1 and Examples 4, 5 and 6, below) or any modification of a material-containing carbon-carbon bond, such as in the production of R-2-hydroxy acid 2 - (indole-3-ylmethyl) -4-ketoglutaric (R-MP) and certain monatin stereoisomers, such as (R, R) - and (S, R) -monatin, and salts thereof.

The polypeptides disclosed herein catalyze the formation carbon-carbon bonds in an aldol reaction and have the ability to use pyruvate or phosphoenolpyruvate as the nucleophilic component in the synthesis of a 4-hydroxy-2-ketobutyrate framework as shown in the scheme general later.

Aldolasa

R = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl R2 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl R3 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl, carboxylic acid.

Without being bound by theory, the conserved four-carbon fragment prepared in all pyruvate aldolase catalyzed condensations is believed to be densely and differentially functionalized. Furthermore, in each adduct, four different oxidation states are contained in four contiguous carbons. The frame

10 prepared by pyruvate aldolases then allows the preparation of α-amino-and-hydroxycarboxylic acids, hydroxycarboxylic acids, α-and-dihydroxycarboxylic acids, and 2-deoxyaldosed sugars as shown in the scheme below.

Therefore, pyruvate aldolases according to some embodiments can be synthetically versatile and

15 can be used in the preparation of a wide range of products for use in animal feed, industrial processes and pharmaceutical agents (see, for example, Gijsen, HJM et al., Recent Advances in the Chemoenzymatic Synthesis of Carbohydrates and Carbohydrate Mimetics, Chem Rev. 1996, 96, 443-473; Henderson, DP et al. J. Org. Chem., Stereospecific Preparation of the N-Terminal Amino Acid Moiety of Nikkomycins KX and KZ via a Multiple Enzyme Synthesis, 1997, 62, 7910 -7911; Wymer, N. & Toone, EJ Enzyme

20 catalyzed Synthesis of Carbohydrates. Current Opin. Chemical Biology, 2000, 4, 110-119).

Polypeptides according to some embodiments may have more than one type of enzyme activity, specifically aldolase activity and additional activity, for example, as set forth in Table 1, below. For example, a polypeptide disclosed herein may have aldolase, pyruvate aldolase activity, HMG and / or KHG aldolase activity. Additionally, the polypeptide may or may be thought to have additional enzymatic activity based on its CE classification. Table 1 includes the column "Expected EC number". A CE number is the number assigned to an enzyme type according to a standardized enzyme nomenclature scheme developed by the Enzyme Commission of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Results in the "Expected EC Number" column are determined by a BLAST search against the Kegg database (Kyoto Encyclopedia of Genes and Genomes). If the maximum BLAST match (also called a "hit") has an E value equal to or less than e − 6, the CE number assigned to the maximum match is entered into the table. CE number

The maximum hit is used as a guide to how the EC number of the sequence of the invention can be. In cases where only a partial CE number is provided, a broad rating could only be assigned based on the maximum hit. For example, in the first row, for SEQ ID NO: 2, coded by SEQ ID NO: 1, the "Expected CE Number is included as" 2. . . " Therefore, the assigned classification is largely a transferase. For SEQ ID NO: 26, encoded by SEQ ID NO: 25, the most specific classification that could

Assigned based on maximum hit is an aldehyde lyase.

Table 1 Source =

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Polypeptides and peptides disclosed herein can be isolated from natural sources, can be synthetic polypeptides, or be generated recombinantly. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides disclosed herein can be made and isolated using any method known in the art. Polypeptides and peptides disclosed herein may also be used, in whole or in part, using chemical methods well known in the art. See for example Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, peptide syntheses can be performed using various solid phase techniques (see for example Roberge (1995) Science 269: 202; Merrifield (1997) Methods Enzymol. 289: 3-13) and automated synthesis can be accomplished, for example using the 431A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

The peptides and polypeptides disclosed herein can also be glycosylated. Glycosylation can be incorporated post-translationally either chemically or through cellular biosynthetic mechanisms, where the latter incorporate the use of known glycosylation motifs, which may be native to the sequence or which may be incorporated as a peptide or added to the sequence of encoding nucleic acids. Glycosylation can be O-linked or N-linked.

In some embodiments, when indicated, the peptides and polypeptides disclosed herein can include all "mimetic" and "peptidomimetic" forms. The terms "mimetics" and "peptidomimetics" refer to a synthetic chemical compound that has basically the same structural and / or functional characteristics of the polypeptides disclosed herein. The mimetic forms may be entirely composed of synthetic, unnatural amino acid analogs or be a chimeric molecule of partially natural peptide amino acids and partially unnatural amino acid analogs. Mimetic forms may also incorporate any number of conservative natural amino acid substitutions to the extent that such substitutions do not also fundamentally alter the structure and / or mimetic activity. As with the polypeptides disclosed herein that are conservative variants or members of a genus of polypeptides disclosed herein (having for example about 50% or more sequence identity with a sequence disclosed herein), the experiments of routine will determine if a mimetic is within the scope disclosed herein, that is, if its structure and / or function is basically not altered. Thus, in some embodiments, a mimetic composition is within the scope of the disclosure if it has an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase.

Polypeptide mimetic compositions disclosed herein can contain any combination of unnatural structural components. In an alternative embodiment, the mimetic compositions disclosed herein include one or all of the following three structural groups: a) residue linking groups other than the natural amide bond ("peptide bond"); b) unnatural residues instead of natural amino acid residues; or c) residues that induce secondary structural mimicry, that is, to induce or stabilize a secondary structure, such as a beta-turn, gamma-turn, beta-sheet, alpha-helix conformation, and the like. For example, a polypeptide disclosed herein can be characterized as a mimetic when all or some of its residues are linked by chemical means other than natural peptide bonds. The individual peptidomimetic residues may be linked by peptide bonds, other chemical bonds, or coupling media, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N'-dicyclohexylcarbodiimide (DCC), or N, N'-diisopropylcarbodiimide (DIC) . Binder groups that may be an alternative to the traditional amide bond ("peptide bond") include, for example, ketomethylene (such as —C (= O) - CH2— by —C (= O) —NH—), aminomethylene (CH2 —NH), ethylene, olefin (CH = CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4—), thiazole, retroamide, thioamide or ester (see Spatola (1983) in Chemistry and Biochemistry from Amino Acids, Peptides and Proteins, Vol. 7, pages 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).

A polypeptide disclosed herein can also be characterized as a mimetic by containing all or some of the residues rather than natural amino acid residues. Unnatural waste is described in the Scientific and Patent Library; Some exemplary unnatural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Aromatic amino acid mimetics can be generated by replacing, for example, D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thienylalanine; D-o L-1, -2, 3- or 4-pyrenylalanine; D- or L-3 thienylalanine; D- or L- (2-pyridinyl) -alanine; D- or L- (3-pyridinyl) alanine; D- or L- (2-pyrazinyl) -alanine; D- or L- (4-isopropyl) -phenylglycine; D- (trifluoromethyl) -phenylglycine; D- (trifluoromethyl) phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D- or L-2indole (alkyl) alanines; and, D- or L-alkylalanines, where the alkyl can be methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, substituted or unsubstituted iso-pentyl or non-acidic amino acids. The aromatic rings of a non-natural amino acid include, for example, aromatic rings of thiazolyl, thiophenyl, porazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl.

Acid amino acid mimetics can be generated by substitution for, for example, non-carboxylate amino acids, keeping a negative charge the same; (phosphono) alanine; sulfated threonine. Carboxyl side groups (such as aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'— N — C — N — R ') such as 1-cyclohexyl-3 (2-morpholinyl- (4-ethyl) carbodiimide or 1-Ethyl-3 (4-azonia-4,4-dimetholpentyl) carbodiimide Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions Basic amino acid mimetics can be generated by substitution with, for example (in addition to lysine and arginine) the amino acids ornithine, citrulline or (guanidino) -acetic acid, or (guanidino) alkyl-acetic acid, where the alkyl is defined above. The nitrile derivative (containing for example the CN moiety instead COOH) can be replaced by asparagine or glutamine. Asparaginyl or glutamyl residues can be deaminated to corresponding aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with for example one or more Conventional reagents, including, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, in some embodiments under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, for example aromatic diazonium compounds or tetranitromethane, N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Mimetics of cysteine residues can be generated by reacting cystyl residues with, for example, alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to provide carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, for example, bromo-trifluoroacetone, alpha-bromo-beta- (5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri4 nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and terminal amino residues can be altered) by reacting lysinyl with, for example, succinic and other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and reactions Transamidase catalyzed with glyoxylate. Methionine mimetics can be generated by reaction with, for example, methionine sulfoxide. Proline mimetics include, for example, pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3, -dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, for example, diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, for example, those generated by hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine, and histidine; acetylation of the N-terminal amine; methylation of the main chain amide residues or substitution with N-methylamino acids; or amidation of the C-terminal carboxyl groups.

In some embodiments, a residue, such as an amino acid, of a polypeptide disclosed herein can be replaced by an amino acid (or peptidomimetic residue) of opposite chirality. In some embodiments, any naturally occurring amino acid in the L configuration (which may also be referred to as R or S, depending on the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of chirality opposite, termed as the D-amino acid, but may also be termed as the R or S form.

The invention also provides methods of modifying the polypeptides disclosed herein by natural processes, such as post-translational processing (such as phosphorylation, acylation, etc.) or by chemical modification techniques, and the resulting modified peptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid chains, and amino termini

or carboxyl. It will be appreciated that the same type of modification may be present to the same degree or to varying degrees at various sites in a given polypeptide. Also a given polypeptide can have many types of modifications. In some embodiments, modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, binding covalent phosphatidylinositol, crosslinking cyclization, disulfide bond formation, demethylation, covalent intermolecular bond formation, cysteine formation, pyroglutamate formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation , pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and RNA mediated addition of amino acid transfer to protein such as arginylation. See Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, p. 1-12 (1983).

Solid phase chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments disclosed herein. Such a method has been known in the art since the early 1960s (Merrifield, RB, J. Am. Chem. Soc., 85: 2149-2154, 1963) (See also Stewart, JM and Young, JD, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., Pp. 11-12)) and was recently used in commercially available laboratory synthesis and design kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally used the findings of H. M. Geysen et al., Proc. Natl. Acad. Sci., USA, 81: 3998 (1984) and provide synthesizing peptides on the tips of multiple "rods" or "pins", all of which are connected to a single plate. When such a system is used, one rod or pin plate is inverted and inserted into a second plate of corresponding wells or reservoirs, containing solutions for attaching or anchoring an appropriate amino acid to the tips of the pins or rods. By repeating said step of the process, that is, by inverting and inserting the tips of the rods or pins into appropriate solutions, the amino acids are incorporated into desired peptides. In addition, several available FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an automated synthesizer from Applied Biosystems, Inc. Model 431A ™. Such equipment provides easy access to the peptides disclosed herein, either by direct synthesis or by synthesis of various fragments that can be coupled using other known techniques.

The polypeptides disclosed herein include aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase in active or inactive form. For example, the polypeptides disclosed herein include proproteins prior to their "maturation" or prepro sequence processing, such as by means of a proprotein processing enzyme, such as a proprotein convertase to generate a "active" mature protein. The polypeptides disclosed herein include aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase inactive for other reasons, such as prior to "activation" by means of a post-translational processing event, such as a action of endo- or exo-peptidase or proteinase, a phosphorylation event, an amidation, a glycosylation or a sulfation, a dimerization event and the like. The polypeptides disclosed herein include all active forms, including active subsequences, such as catalytic domains or active sites, of the enzyme.

The invention includes immobilized aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, anti-aldolase, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase antibodies and fragments thereof . In some embodiments, the invention provides methods of inhibiting the activity of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, for example using dominant negative mutants or anti-aldolase, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase antibodies disclosed herein. Heterocomplexes, such as fusion proteins, heterodimers, etc., comprising the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein, are also disclosed.

In some embodiments, the polypeptides disclosed herein may have aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, under different conditions, such as at extreme pH and / or temperature, or, in some embodiments , in the presence of oxidizing agents. In some embodiments, the disclosure provides methods that produce the alternative aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase preparations with different catalytic efficiencies and stabilities, such as toward temperature, oxidizing agents, and changing washing conditions. In some embodiments, variants of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be produced using targeted mutagenesis and / or random mutagenesis techniques. In some embodiments, targeted evolution can be used to produce a wide variety of aldolase enzyme, such as pyruvate aldolase enzyme variants, such as HMG and / or KHG aldolase with alternative specificities and stability.

The proteins disclosed herein are also useful as research reagents for identifying aldolase enzyme modulators, such as pyruvate aldolase, such as HMG and / or KHG aldolase, such as activators or inhibitors of aldolase enzyme activity, such as pyruvate aldolase , such as HMG and / or KHG aldolase. Briefly, test samples (compounds, broths, extracts, and the like) are incorporated into aldolase enzyme assays, such as pyruvate aldolase, such as HMG and / or KHG aldolase to determine their ability to inhibit substrate cleavage. Inhibitors identified in this way can be used in industry and in research to reduce or prevent unwanted proteolysis. As with aldolase, such as pyruvate aldolase enzymes, such as HMG and / or KHG aldolases, inhibitors can be combined to increase the spectrum of activity.

The enzymes disclosed herein are also useful as research reagents for digesting proteins or for protein sequencing. For example, aldolase, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase can be used to break polypeptides into smaller fragments for sequencing using, for example, an automated sequencer.

The invention also provides methods for discovering a new aldolase, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase using the nucleic acids, polypeptides and antibodies disclosed herein. In some embodiments, phagemid libraries are screened for discovery based on aldolase expression, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase. In other embodiments, lambda phage libraries are screened for discovery based on expression of aldolase, such as pyruvate aldolase enzyme, such as HMG and / or KHG aldolase. Evaluation of phage or phagemid libraries may allow detection of toxic clones; better access to the substrate; reduced need to manipulate a host, avoid the possibility of any bias resulting from massive deletion of the library; and, faster growth at low clone densities. The evaluation of phage or phagemid libraries can be in liquid phase or solid phase. In some embodiments, the invention provides liquid phase evaluation. This provides greater flexibility under test conditions; additional substrate flexibility; highest sensitivity for weak clones; and ease of automation during solid phase evaluation.

Evaluation methods using the proteins and nucleic acids disclosed herein and robotic automation are also described to allow thousands of biocatalytic reactions and detection assays to be run in a short period of time, such as per day, and to ensure a high level of precision and reproducibility (see the description of the arrangements, below). As a result, a library of derived compounds can be produced in a matter of weeks. For more information on modifying molecules, including small molecules, see PCT / US94 / 09174; United States Patent No. 6,245,547.

In some embodiments, the polypeptides or fragments disclosed herein are obtained through the biochemical enrichment or purification procedures. The sequence of homologous polypeptides or fragments can be determined by aldolase enzyme assays, such as pyruvate aldolase, such as HMG and / or KHG aldolase (see Examples 3, 4 and 5, below), gel electrophoresis and / or microsequencing. The sequence of the possible polypeptide or fragment disclosed herein can be compared to a polypeptide disclosed herein or a fragment, such as comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75 , 100 or 150 or more consecutive amino acids thereof using any of the programs described above.

An assay is also disclosed to identify fragments or variants disclosed herein, which retain the enzymatic function of the polypeptides disclosed herein. For example, fragments or variants of such polypeptides can be used to catalyze biochemical reactions, indicating that the fragment or variant retains the enzymatic activity of a polypeptide disclosed herein. An example of an assay to determine if variant fragments retain the enzymatic activity of the polypeptides disclosed herein includes the steps of: contacting the polypeptide or variant fragment with a substrate molecule under conditions that allow the fragment of the polypeptide or variant work and detect a decrease in the level of substrate

or an increase in the level of the specific reaction product of the reaction between the polypeptide and substrate.

The present disclosure exploits the unique catalytic properties of enzymes. While the use of biocatalysts (i.e., purified or crude enzymes, living or non-living cells) in chemical transformations normally requires the identification of a particular biocatalyst that reacts with a specific starting compound, the present method uses selected biocatalysts and conditions of reaction that are specific for functional groups that are present in many starting compounds, such as small molecules. Each biocatalyst is specific for one functional group or several related functional groups and can react with many starting compounds that contain this functional group.

In some embodiments, biocatalytic reactions produce a population of derivatives of a single starting compound. These derivatives can undergo another round of biocatalytic reactions to produce a second population of derived compounds. Thousands of variations of the original small molecule or compound can occur with each iteration of biocatalytic derivatization.

Enzymes react at specific sites in a starting compound without affecting the rest of the molecule, a process that is difficult to accomplish using traditional chemical methods. This high degree of biocatalytic specificity provides the means to identify a single active compound within the library. The library is characterized by the series of biocatalytic reactions used to produce it, a so-called "biosynthetic history". Evaluation of the library for biological activities and the location of the biosynthetic history identifies the specific reaction sequence that the active compound produces. The reaction sequence is repeated and the structure of the synthesized compound determined. This mode of identification, unlike other synthesis and detection approaches, does not require immobilization technologies and the compounds can be freely synthesized and evaluated in solution using virtually any type of detection assay. It should be noted that the high degree of specificity of enzymatic reactions in functional groups allows the “localization” of specific enzymatic reactions that form the biocatalytically produced library.

In some embodiments, the process steps are performed using robotic automation that allows thousands of biocatalytic reactions and / or detection assays to be run per day as well as ensuring a high level of precision and reproducibility. Robotic automation can also be used to assess aldolase activity to determine if a polypeptide is within the scope of the disclosure. As a result, in some embodiments, a library of derived compounds can be produced in a matter of weeks that would take years to produce using "traditional" chemical or enzymatic detection methods.

Further disclosed are methods of modifying small molecules, which comprise contacting a polypeptide encoded by a polynucleotide described herein or enzymatically active fragments thereof with a small molecule to produce a modified small molecule. A library of modified small molecules is evaluated to determine if a modified small molecule is present in the library, which exhibits desired activity. A specific biocatalytic reaction that produces the modified small molecule of desired activity by systematically eliminating each of the biocatalytic reactions used to produce a portion of the library and then evaluating the small molecule produced in the portion of the library to determine the presence or absence of the small molecule modified with the desired activity. Specific biocatalytic reactions that produce the modified small molecule of desired activity are optionally repeated. Biocatalytic reactions are carried out with a group of biocatalysts that react with different structural residues that are within the structure of a small molecule, each of the biocatalysts is specific for a structural residue or a group of related structural residues; and each of the biocatalysts reacts with many different small molecules that contain the different structural moiety.

Aldolase enzyme signal sequences, such as pyruvate aldolase, such as HMG and / or KHG aldolase, prepro and catalytic domains

The disclosure provides aldolase enzyme signal sequences, such as pyruvate aldolase, such as HMG and / or KHG aldolase (such as signal peptides (SP)), prepro domains, and catalytic domains (CD). The SPs, prepro and / or CD domains disclosed herein can be isolated, synthetic, or recombinant peptides or can be part of a fusion protein, such as a heterologous domain in a chimeric protein. Nucleic acids encoding these catalytic domains (CD), prepro domains, and signal sequences (SP, such as a peptide having a sequence comprising / consisting of amino terminal residues of a polypeptide disclosed herein) are also described.

The invention provides isolated, synthetic or recombinant signal sequences (such as signal peptides) consisting of or comprising a sequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 46 or 1 to 47, of a polypeptide disclosed herein, such as polypeptides disclosed herein, see also Table 1, Examples 4, 5 and 6, below, and the Sequence Listing. For example, Table 1, above, sets forth exemplary signal (leader) sequences disclosed herein, such as in the polypeptide having a sequence as set forth in SEQ ID NO: 66, encoded, for example, by SEQ ID NO. : 65, has a signal sequence comprising (or consisting of) the 27 amino terminal residues or MSIVVTKIERAGAAAVAALRTSGVATV (SEQ ID NO: 407) corresponding to the first 27 amino acids of SEQ ID NO: 66.

There are signal sequences that comprise the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino terminal residues of a polypeptide disclosed herein.

There are polypeptides with or without a signal sequence and / or a prepro sequence. In some embodiments, there are polypeptides with heterologous signal sequences and / or prepro sequences. The prepro sequence (including a sequence disclosed herein used as a heterologous prepro domain) can be located at the amino terminus

or carboxy protein. There are also isolated, synthetic or recombinant sequence signals, prepro sequences, and catalytic domains (such as "active sites") that comprise sequences disclosed herein. The polypeptide comprising a signal sequence disclosed herein can be an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, disclosed herein or another aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase or another enzyme or another polypeptide. Methods for identifying "prepro" domain sequences and signal sequences are well known in the art, see Van de Ven (1993) Crit. Rev. Oncog. 4 (2): 115-136. For example, to identify a prepro sequence, the protein is purified from the extracellular space, and the sequence of the N-terminal protein is determined and compared to the raw form.

The signal sequences (SP) and / or prepro sequences of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein can be isolated, synthetic or recombinant peptides, or sequences linked to another aldolase enzyme , such as pyruvate aldolase, such as HMG and / or KHG aldolase or a non-aldolase, such as non-pyruvate aldolase, eg, non-HMG and / or non-KHG aldolase polypeptide, such as a fusion protein (chimeric). Further disclosed are polypeptides comprising aldolase enzyme signal sequences, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein. In some embodiments, polypeptides comprising SP signal and / or aldolase enzyme prepro, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein comprise heterologous sequences to an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, disclosed herein (such as a fusion protein comprising an SP and / or prepro disclosed herein and sequences of another aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase or a non-aldolase, such as non-pyruvate aldolase, eg, non-HMG protein and / or non-KHG aldolase). Aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein with SP and / or heterologous prepro sequences, such as sequences with a signal yeast signal sequence, are also disclosed. An aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase disclosed herein can comprise a heterologous SP and / or prepro in a vector, such as a pPIC series vector (Invitrogen, Carlsbad, CA).

In some embodiments, the SP and / or prepro sequences disclosed herein are identified by following an identification of novel aldolase peptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The pathways by which proteins are classified and transported to their proper cellular location are often called pathways that target proteins. One of the most important elements in all of these targeting systems is a short amino acid sequence at the amino terminus of a recently synthesized polypeptide called the signal sequence. This signal sequence directs a protein to its proper location in the cell and is removed during transport or when the protein reaches its final destination. Most of the lysosomal, membrane or secreted proteins have an amino-terminal signal sequence that marks the translocation towards the lumen in the endoplasmic reticulum. The length of the signal sequences can vary from about 10 to 65, or more, amino acid residues. Various methods for signal sequence recognition are known to those of skill in the art. For example, in some embodiments, signals from novel aldolase enzyme peptides, such as pyruvate aldolase, such as HMG and / or KHG aldolase, are identified by a method called SignalP. SignalP uses a combined neural network that recognizes both peptide signals and their cleavage sites. (Nielsen (1997) “Identification of prokaryotic and eukaryotic peptides signals and prediction of their cleavage sites.” Protein Engineering 10: 1-6.

In some embodiments, aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase according to the invention do not have prepro SP sequences and / or "domains". Also disclosed is the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein that lacks all or part of a SP and / or prepro domain. Nucleic acid sequences encoding a signal (SP) and / or prepro sequence of an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase operatively linked to a nucleic acid sequence of a different aldolase, such may be desired. as the pyruvate aldolase enzyme, such as HMG and / or KHG aldolase or optionally a signal sequence (SP) and / or prepro domain of a non-aldolase, such as non-pyruvate aldolase, for example, a non-HMG and / or non-KHG aldolase protein .

Isolated, synthetic or recombinant polypeptides comprising signal sequences (SP), prepro domain and / or catalytic domains (CD) disclosed herein and heterologous sequences are also disclosed. Heterologous sequences are sequences that are not naturally associated (such as to an enzyme) with a SP, prepro domain and / or CD. The sequence to which the SP, the prepro domain and / or CD do not naturally associate can be in the SP, prepro domain and / or amino terminal end of CD, carboxy terminal end and / or both ends in the SP and / or CD. In some embodiments, the invention provides isolated, synthetic, or recombinant polypeptides that comprise (or consist of) a polypeptide that comprises a signal sequence (SP), a prepro domain, and / or a catalytic domain (CD) disclosed herein with the provided that they are not associated with any sequence to which they are not naturally associated (such as an aldolase enzyme, such as a pyruvate aldolase, HMG and / or KHG aldolase sequence). Similarly, there are isolated, synthetic, or recombinant nucleic acids that encode these polypeptides. Thus, in some embodiments, the isolated, synthetic or recombinant nucleic acid disclosed herein comprises a coding sequence for a signal sequence (SP), a prepro domain and / or a catalytic domain (CD) disclosed herein and a sequence heterologous (i.e., a sequence that is not naturally associated with a signal sequence (SP), a prepro domain and / or catalytic domain (CD) disclosed herein). The heterologous sequence may be at the 3 'end, the 5' end and / or at both ends of the SP, the prepro domain and / or the CD coding sequence.

Hybrid (chimeric) aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolases and peptide libraries

In some embodiments, the invention provides a hybrid aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, and fusion proteins, including peptide libraries, comprising sequences disclosed herein. The peptide libraries disclosed herein can be used to isolate modular peptides (such as activators or inhibitors) from targets, such as substrates, receptors, enzyme aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The peptide libraries disclosed herein can be used to identify formal binding parts, such as ligands, such as cytokines, hormones, and the like. In some embodiments, the invention provides chimeric proteins comprising a signal sequence (SP), a prepro domain and / or a catalytic domain (CD) disclosed herein or a combination thereof and a heterologous sequence (see above).

In some embodiments, the fusion proteins disclosed herein (such as the peptide moiety) are conformationally stabilized (relative to linear peptides) to allow for higher binding affinity for targets. Aldolase enzyme fusions, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein and other peptides, including known peptides and random peptides, are further described. They can be fused in such a way that the structure of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolases is not considerably disturbed and that the conformation of the peptide is metabolically or structurally stabilized. This allows the creation of a library of peptides that is easily monitored to determine their presence in cells and their quantity.

Variants of amino acid sequences disclosed herein can be characterized by a predetermined nature of the variation, a characteristic that separates them naturally, such as an allelic or interspecies variation of an aldolase enzyme, such as a pyruvate aldolase sequence, HMG and / or KHG aldolase. In some embodiments, the variants disclosed herein exhibit the same qualitative biological activity as the natural analog. Alternatively, variants can be selected for having modified characteristics. In some embodiments, while the site or region for introducing a variation of the amino acid sequence is predetermined, the mutation itself does not need to be determined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be carried out on the target codon or region and the expressed aldolase enzyme variants, such as pyruvate aldolase, such as HMG and / or KHG aldolase evaluated for the optimal combination of the desired activity. Techniques for making such substitution mutations at predetermined sites in DNA having a known sequence are well known, as described for example herein, M13 primer mutagenesis and PCR mutagenesis. Mutant evaluation can be performed using, for example, carbon-carbon bond formation or cleavage assays. In other embodiments, the amino acid substitutions can be single residues; insertions can be made in the order of about 1 to 20 amino acids, although more insertions can be made. Eliminations can be in the range of about 1 to about 20, 30, 40, 50, 60, 70 residues

or more. To obtain a final derivative with optimal properties, substitutions, deletions, inserts, or any combination thereof may be used. Generally, these changes are made in a few amino acids to minimize disruption of the molecule. However, greater changes may be tolerated in certain circumstances.

Aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase exists where the central structure of the polypeptide, the secondary structure or the tertiary structure, such as an alpha helix or beta sheet structure, has been modified. In some embodiments, the charge or hydrophobicity has been modified. In some embodiments, the volume of a side chain has been modified. Significant changes in immune function or identity are obtained by selecting substitutions that are less conservative. For example, substitutions may be made that more significantly affect: the structure of the core structure of the polypeptide in the area of alteration, for example an alpha helix or beta sheet structure; a charges a hydrophobic site on the molecule, which may be at an active site; or a side chain. Further disclosed are substitutions in polypeptides disclosed herein where (a) hydrophilic residues, such as seryl or threonyl, are replaced by a hydrophobic residue, such as leucyl, isoleucil, phenylalanyl, valine or alanyl; (b) a cysteine or proline is replaced by any other residue;

(c)

 a residue having an electropositive side chain, such as lysyl, arginyl, or histidyl, is replaced by an electronegative residue, such as glutamyl or aspartyl; or (d) a residue that has a bulky side chain, such as phenylalanine, is replaced by one that does not have a side chain, such as glycine. Variants can exhibit the same qualitative biological activity (i.e., an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase) although variants can be selected to modify the characteristics of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolases, as needed.

In some embodiments, the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein comprises purification tags or epitopes, signal sequences, or other fusion sequences, etc. In some embodiments, the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein can be fused with a random peptide to form a fusion polypeptide. "Fused" or "operably linked" herein means that the random peptide and the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase are bound, so as to minimize disruption of enzyme stability aldolase, such as pyruvate aldolase, such as a HMG and / or KHG aldolase structure, which for example retains the activity of aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase. The fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) may also comprise other components, including multiple peptides in multiple rings.

In some embodiments, the peptides and nucleic acids encoding them are randomized, either completely randomized or skewed in their randomization, such as nucleotide / residue frequency generally or by position. "Randomized" means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. In some embodiments, the nucleic acids that give rise to the peptides can be chemically synthesized, and therefore can incorporate any nucleotide at any position. Thus, when nucleic acids are expressed to form peptides, any amino acid residue can be incorporated at any position. The synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the combinations in the length of the nucleic acid, thus forming a library of randomized nucleic acids. The library can provide a population with a sufficiently diverse structure of randomized expression products to influence a range of likely cellular responses sufficient to provide one or more cells that exhibit a desired response. In this way, large enough interaction libraries are disclosed so that at least one of the members will have a structure that gives it affinity for some molecules, proteins or other factors.

In some embodiments, an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase, disclosed herein is a multi-domain enzyme comprising a peptide signal, a carbohydrate binding module, an aldolase enzyme, such as a Pyruvate aldolase, HMG and / or KHG aldolase catalytic domain, a binder and / or other catalytic domain.

The invention provides methods and sequences for generating chimeric polypeptides that can encode biologically active hybrid polypeptides (such as a hybrid aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase). In some embodiments, the original polynucleotides (such as a nucleic acid disclosed herein) encode biologically active polypeptides. In some embodiments, a method disclosed herein produces novel hybrid polypeptides using cellular processes that integrate the sequence of the original polynucleotides such that the resulting hybrid polynucleotide encodes a polypeptide that demonstrates activities derived from, but different from, biologically active polypeptides. originals (such as aldolase or antibody according to the invention). For example, the original polynucleotides can encode a particular enzyme (such as aldolase) from or found in different microorganisms, an enzyme that encodes a first polynucleotide of an organism or variant can, for example, function effectively in an environmental condition In particular, such as high salinity, an enzyme encoding a second polynucleotide from a different or variant organism can function effectively under a different environmental condition, such as extremely high temperatures. A hybrid polynucleotide containing sequences of the original first and second polynucleotides can encode an enzyme that exhibits characteristics of both enzymes encoded by the original polynucleotides. Thus, the enzyme encoded by the hybrid polynucleotide disclosed herein can function effectively under ambient conditions shared by each of the enzymes encoded by the first and second polynucleotides, such as high salinity and extreme temperatures.

In some embodiments, a hybrid polypeptide generated by a method disclosed herein can exhibit a specialized enzyme not exhibited in the original enzymes. For example, by following the recombination and / or reductive rearrangement of polynucleotides encoding enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolases, the resulting hybrid polypeptide encoded by a hybrid polynucleotide can be screened for nonenzyme activity specialized aldolase, such as non-pyruvate aldolase, such as non-HMG and / or non-KHG-aldolase enzyme, such as hydrolase, peptidase, phosphorylase, etc. activities, obtained from each of the original enzymes. In some embodiments, the hybrid polypeptide is evaluated to determine such chemical functionalities that distinguish the hybrid polypeptide from the original base polypeptides, such as the temperature, pH, or salt concentration at which the hybrid polypeptide functions.

Also described is a method of producing a biologically active hybrid polypeptide and evaluating said polypeptide for enhanced activity:

•

1) introducing at least a first polynucleotide linkage operative and a second polynucleotide linkage operative, at least the first and second polynucleotides sharing at least one region of partial homology, into a suitable host cell;

•

2) culturing the host cell under conditions that promote sequence rearrangement resulting in an operably linked hybrid polynucleotide;

•

3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide;

•

4) evaluating the hybrid polypeptide under conditions that promote the identification of enhanced biological activity; and

•

5) isolating a polynucleotide that encodes the hybrid polypeptide.

Isolate and discover aldolase enzymes

There are methods for isolating and discovering aldolase, such as pyruvate aldolase, HMG and / or KHG aldolase enzymes and the nucleic acids that encode them. Polynucleotides or enzymes can be isolated from individual organisms ("isolates"), pools of organisms that have been cultured in defined media ("enrichment cultures"), or uncultivated organisms ("environmental samples"). Organisms can be isolated, for example, by in vivo affinity selection (biopanning) cycles (see details below). Using a culture-independent approach to derive polynucleotides that encode novel bioactivities from environmental samples is more preferable because it allows access to untapped biodiversity resources. Polynucleotides or enzymes can also be isolated from any one of numerous organisms, such as bacteria. In addition to whole cells, polynucleotides or enzymes can also be isolated from crude enzyme preparations derived from cultures of these organisms, such as bacteria.

"Environmental libraries" are generated from environmental samples and represent the collective genomes of natural organisms obtained by cloning vectors that can be propagated into suitable prokaryotic hosts. Since cloned DNA is initially extracted directly from environmental samples, libraries are not limited to the small fraction of prokaryotes that can be grown in a pure culture. Furthermore, a normalization of the environmental DNA present in these samples could allow a more equitable representation of the DNA of all the species present in the original sample. This can dramatically increase the efficiency of the search for interesting genes from minor constituents of the sample that may be represented in orders of magnitude much lower compared to the dominant species.

In some embodiments, gene libraries generated from one or more uncultivated microorganisms are routinely detected for an activity of interest. Possible pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries. In some embodiments, polynucleotides encoding activities of interest are isolated from said libraries and introduced into a host cell. The host cell is cultured under conditions that promote reductive recombination and / or rearrangement, creating potentially active biomolecules with novel or improved activities.

In vivo affinity selection can be performed using FACS-based and non-optical (such as magnetic) machines. In some embodiments, complex gene libraries are constructed with vectors containing elements that stabilize the transcribed RNA. For example, the inclusion of sequences that result in secondary structures such as hairpins, which are designed to flank the transcribed regions of the RNA, would serve to improve its stability, thereby increasing its half-life within the cell. The probe molecules used in the affinity selection cycle process consist of reporter molecule-labeled oligonucleotides that only fluoresce upon probe binding to a target molecule. These probes are introduced into the recombinant cells in the library using one of several transformation methods. The molecules in the probes bind to the transcribed target mRNA, resulting in heteroduplex DNA / RNA molecules. Linking the probe to a target will yield a fluorescent signal that is detected and pooled by the FACS machine during the systematic screening process.

In some embodiments, subcloning is performed to isolate even more sequences of interest. In subcloning, a portion of DNA is amplified and digested, generally by restriction enzymes, to cut the desired sequence, which binds to a receptor vector and is amplified. At each stage of subcloning the portion is examined for the activity of interest in order to ensure that DNA encoding the structural protein has not been excluded. The insert can be purified at any stage of subcloning, for example, by gel electrophoresis before ligation into the vector or where cells that contain the receptor vector and cells that do not contain the receptor vector are placed in selective medium that it contains, for example, an antibiotic that will kill cells that do not contain the receptor vector. Specific methods of subcloning cDNA inserts into vectors are well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)). In other embodiments, the enzymes disclosed herein are subclones. These subclones may differ from the base clone, for example, in length, a mutation, or a tag.

Microorganisms from which the polynucleotide can be discovered, isolated, or prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae, and protozoa. Polynucleotides can be discovered, isolated, or prepared from environmental samples, in which case the nucleic acid can be recovered without culturing an organism or recovered from one or more cultured organisms. In some embodiments, said microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychotrophs, halophiles, barophiles, and acidophiles. Polynucleotides encoding enzymes isolated from extremophilic microorganisms can be used. The enzymes of the present invention can function at temperatures above 100 ° C, such as those found in terrestrial hot springs and underwater thermal vents, or at temperatures below 0 ° C, such as those found at Arctic waters, in a saturated saline environment, such as those found in the Dead Sea, at pH values close to 0, such as those found in coal deposits and sulfur-rich geothermal springs, or at higher pH values to 11, such as those found in sewage sludge. In some embodiments, the enzymes disclosed herein have high activity over a wide range of temperatures and pH values.

The selected and isolated polynucleotides as described above are introduced into a suitable host cell. A suitable host cell is any cell that is capable of promoting recombination and / or reductive rearrangement. In some embodiments, the selected polynucleotides are already in a vector that includes suitable control sequences. The host cell may be an upper eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or, in some embodiments, the host cell may be a prokaryotic cell, such as a bacterial cell. The introduction of the construct into the host cell can be carried out by transfection with calcium phosphate, DEAE-Dextran-mediated transfection or electroporation.

Examples of hosts include bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS, or Bowes melanoma; adenovirus; and plant cells; see details above. Selection of an appropriate host is considered to be within the scope of those skilled in the art from the teachings herein.

Various mammalian cell culture systems can be employed to express recombinant protein; examples of mammalian expression systems include the monkey kidney fibroblast COS-7 lines described in "SV40-transformed simian cells support the replication of early SV40 mutants" (Gluzman, 1981) and other cell lines capable of expressing a compatible vector for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors can comprise a suitable origin of replication, a promoter and enhancer and also ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5 'flanking non-transcribed sequences that are needed. DNA sequences derived from the SV40 junction and polyadenylation sites can be used to provide the necessary untranscribed genetic elements.

In other embodiments, nucleic acids, polypeptides, and methods disclosed herein are used in biochemical pathways or to generate novel polynucleotides encoding biochemical pathways of one or more operons or gene clusters thereof. For example, bacteria and many eukaryotes have a coordinated mechanism to regulate genes whose products participate in related processes. Genes are grouped into structures called "gene clusters" on a single chromosome and transcribed together under the control of a single regulatory sequence, including a single promoter that initiates transcription of the entire cluster. Thus, a gene cluster is a group of adjacent genes that are identical or related, generally in terms of their function (an example of a biochemical pathway encoded by a gene cluster is polyketides).

In some embodiments, the DNA from the gene cluster is isolated from different organisms and ligated into vectors, such as vectors that contain expression regulatory sequences that can control and regulate the production of a protein activity or related protein array detectable by linked gene clusters. The use of vectors that have an exceptionally large capacity for the introduction of exogenous DNA may be appropriate for use with such gene pools and are described by way of example herein to include factor f (or fertility factor) from E. coli. This E. coli factor f is a plasmid that affects high-frequency transfer of itself during conjugation and is ideal for obtaining and stably propagating large pieces of DNA, such as gene pools from mixed microbial samples. In one embodiment, cloning vectors, called "fossils" or bacterial artificial chromosome (BAC) vectors are used. These are derived from E. coli factor f, which is capable of stably integrating large segments of genomic DNA. When integrated with the DNA of a mixed environmental sample it is possible to obtain large genomic fragments in the form of a stable "environmental DNA library". Another type of vector for use is a cosmid vector. Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA. Cloning into cosmid vectors is described in detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated to an appropriate vector, two or more vectors containing different groupings of polyketide synthase genes can be introduced into a suitable host cell. Regions of partial sequence homology shared by gene clusters will promote processes that result in sequence rearrangement, resulting in a hybrid gene cluster. Subsequently, the novel hybrid gene cluster can be systematically analyzed to detect improved activities that were not found in the original gene clusters.

Methods for systematically detecting various enzyme activities are known to those skilled in the art and are described herein (see Examples 1, 2 and 3 below. These methods can be used when isolating the polypeptides and polynucleotides disclosed in the present.

There are methods for discovering and isolating aldolases, such as pyruvate aldolase, such as HMG and / or KHG aldolase,

or compounds to modify the activity of these enzymes, using a whole cell approach (see description below). Putative aldolase-encoding clones, such as pyruvate aldolase, such as HMG and / or KHG aldolase, can be systematically detected from genomic DNA libraries.

Automatic detection methodologies and "online" monitoring devices

In the implementation of the methods disclosed herein, various apparatus and methodologies may be used in conjunction with the polypeptides and nucleic acids disclosed herein, for example, to systematically detect polypeptides for enzyme aldolase activity, such as pyruvate aldolase, such such as HMG and / or KHG aldolase, to systematically detect compounds as possible modulators, such as activators

or inhibitors, of an aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, for antibodies that bind to a polypeptide disclosed herein, for nucleic acids that hybridize to a nucleic acid disclosed herein. present, to systematically detect cells expressing a polypeptide disclosed herein and the like. In addition to the various formats described in detail below for screening samples systematically, alternative formats can also be used to implement the methods disclosed herein. These formats include, for example, mass spectrometers, chromatographs, such as high-performance HPLC and other forms of liquid chromatography, and smaller formats, such as 1536-well plates, 384-well plates, etc. High performance screening devices can be adapted and used to implement the methods disclosed herein. See United States Patent Applications Nos. 20020001809 and 20050272044.

Capillary arrangements

The nucleic acids or polypeptides disclosed herein can be immobilized or applied to an array. The arrays can be used to systematically detect or monitor libraries of compositions (such as small molecules, antibodies, nucleic acids, etc.) for their ability to bind or modulate the activity of a nucleic acid or polypeptide disclosed herein. Capillary arrays, such as the GIGAMATRIX ™, Diversa Corporation, San Diego, Calif .; and the arrangements described, for example, in United States Patent Application No. 20020080350 A1; WO 0231203 A; WO 0244336 A, provide an alternative apparatus for systematically holding and detecting samples. In some embodiments, the capillary array includes a plurality of capillaries that form an adjacent capillary array, where each capillary comprises at least one lumen-defining wall to retain a sample. The lumen can be cylindrical, square, hexagonal or have any other geometric shape, as long as the walls form a lumen for the retention of a liquid or sample. The capillaries in the capillary array can be held together at a very close distance to form a flat structure. The capillaries can be joined, for example fused (such as when the capillaries are made of glass), glued, joined or fixed side by side. In addition, the capillary array may include interstitial material placed between adjacent capillaries in the array to form a solid flat device containing a plurality of through holes.

A Capillary Array can be formed with any number of individual capillaries, for example, in a range of 100 to 4,000,000 capillaries. Furthermore, a Capillary Array that has approximately 100,000 individual capillaries or more can be given the standard size and shape of a Microtiter® plate to fit standard laboratory equipment. The lumens are filled manually or automatically using capillary action or microinjection using a thin needle. Samples of interest can then be removed from the individual capillaries for further analysis or characterization. For example, a thin needle-like probe is placed in fluid communication with a selected capillary to add or remove material from the lumen.

In a one-step screening test, the components of the assay are mixed to give a solution of interest prior to insertion into the capillary array. The lumen is filled by capillary action when at least a portion of the array is immersed in a solution of interest. Chemical or biological reactions and / or activity in each capillary are monitored for detectable events. Often, a detectable event is called a "hit" that can usually be distinguished from capillaries that produce a "no hit" by optical detection. In this way, the capillary arrays allow enormous parallel detection of "hits".

In a multi-step screening test, a polypeptide or nucleic acid, such as a ligand, can be introduced into a first component, which is introduced into at least a portion of a capillary or capillary array. Then an air bubble can be introduced into the capillary behind the first component. Subsequently, a second component can be introduced into the capillary, where the second component is separated from the first component by the air bubble. The first and second components can then be mixed by applying hydrostatic pressure on both sides of the capillary arrangement to collapse the bubble. The capillary arrangement can then be monitored for a detectable event that results from the reaction or non-reaction of the two components.

In a binding screening test, a sample of interest may be introduced as a first liquid labeled with a detectable particle into a capillary or capillary arrangement, where the capillary lumen is coated with a binding material to bind the detectable particle to the lumen. The first liquid can then be withdrawn from the capillary, whereby the attached detectable particle is held within the capillary, and a second liquid can subsequently be introduced into the capillary. The capillary can then be monitored for a detectable event that results from the reaction or non-reaction of the particle with the second liquid.

Fixes or "Biochips"

The nucleic acids or polypeptides disclosed herein can be immobilized or applied to an array. The arrays can be used to systematically detect or monitor libraries of compositions (such as small molecules, antibodies, nucleic acids, etc.) for their ability to bind or modulate the activity of a nucleic acid or polypeptide disclosed herein. For example, a monitored parameter is transcript expression of an aldolase enzyme gene, such as pyruvate aldolase, HMG, and / or KHG aldolase. One or more or all of the transcripts in a cell can be measured by hybridization of a sample comprising cell transcripts or nucleic acids representative of a cell or complementary to transcripts of a cell, by hybridization with nucleic acids immobilized in an array or "biochip". . " By using a nucleic acid "array" on a microchip it is possible to simultaneously quantify some or all of the transcripts. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a recently engineered strain obtained by the methods disclosed herein. "Polypeptide arrays" can also be used to simultaneously quantify a plurality of proteins. The present method can be practiced with any known "array", also called a "microarray" or "nucleic acid array" or "polypeptide array" or " antibody array "or" biochip ", or a variation thereof. Arrays are generically a plurality of" sites "or" target elements ", each target element comprising a defined amount of one or more biological molecules, such as oligonucleotides, immobilized on a defined area of a substrate surface for specific binding to a sample molecule, such as mRNA transcripts.

The terms "array" or "microarray" or "biochip" or "chip", as used herein, are a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized on a defined area of a substrate surface, as detailed below.

In practicing the methods disclosed herein, any arrangement and / or method for manufacturing and using arrangements in whole or in part, or variations thereof, may be incorporated as described, for example, in United States Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also other documents, such as WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also other publications, such as Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20: 399-407; Bowtell (1999) Nature Genetics Supp. 21: 25-32. See also United States Patent Applications Published Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.

Antibodies and antibody-based screening methods

There are isolated, synthetic or recombinant antibodies that specifically bind to an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase, disclosed herein. These antibodies can be used to isolate, identify, or quantify aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope of the disclosure or other related aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase. Antibodies can be designed to bind to an active site of an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase. Thus, there are methods to inhibit aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase using the antibodies disclosed herein (see description above regarding applications for anti-aldolase enzyme compositions, such as anti-pyruvate aldolase, such as anti-HMG and / or anti-KHG aldolase disclosed herein).

The term "antibody" includes a peptide or polypeptide derived or modeled from, or encoded by, an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding to an antigen or epitope, see Fundamental Immunology, Third Edition, WE Paul, ed., Raven Press, NY (1993); Wilson (1994) J. Immunol. Methods 175: 267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25: 85-97. The term antibody includes portions that bind to antigens, ie, "antigen binding sites" (such as fragments, subsequences, complementarity determining regions (CDRs)) that retain the ability to bind to the antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the single arm VL and VH domains of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), consisting of a domain VH; and (vi) an isolated Complementarity Determining Region (CDR). Single chain antibodies are also included by reference in the term "antibody".

There are fragments of the enzymes disclosed herein (such as peptides), including immunogenic fragments (such as subsequences) of a polypeptide disclosed herein. There are also compositions comprising a polypeptide or peptide disclosed herein and adjuvants or carriers and the like.

Antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like. If desired, nucleic acid sequences encoding specific antigens can be generated by immunization with subsequent isolation of polypeptide or nucleic acid, amplification or cloning, and immobilization of polypeptide on an array disclosed herein. Alternatively, the methods disclosed herein can be used to modify the structure of an antibody produced by a cell to be modified, such as increasing or decreasing the affinity of an antibody. Furthermore, the ability to make or modify antibodies can be a phenotype manipulated and introduced into a cell by the methods disclosed herein.

Antibody immunization, production and isolation methods (polyclonal and monoclonal) are known to those skilled in the art and are described in the scientific and patent literature; see Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256: 495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies can also be generated in vitro, such as by using recombinant antibody binding sites that express phage display libraries, in addition to traditional in vivo methods using animals. See Hoogenboom (1997) Trends Biotechnol. 15: 62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26: 27-45.

The polypeptides disclosed herein or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or 150 consecutive amino acids thereof can also be used to generate antibodies that specifically bind to the polypeptides or fragments. The resulting antibodies can be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine if the polypeptide is present in a biological sample. In these procedures, a protein preparation, such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the peptides disclosed herein, or fragments comprising at least 5, 10, 15 , 20, 25, 30, 35, 40, 50, 75, 100 or 150 consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solid support, such as a bead or other column matrix. The protein preparation is placed in contact with the antibody under conditions where the antibody specifically binds to one of the polypeptides disclosed herein or a fragment thereof. After washing to remove non-specifically bound proteins, specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibody can be determined using various procedures known to those of skill in the art. For example, binding can be determined by labeling the antibody with a detectable label, such as a fluorescent agent, an enzyme label, or a radioisotope. Alternatively, binding of the antibody to the sample can be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies raised against the polypeptides disclosed herein, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, eg, a non-human animal. The thus obtained antibody can bind to the polypeptide. In this way, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies that can bind to the entire native polypeptide. Such antibodies can be used to isolate the polypeptide from cells that express that polypeptide.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by cultures of continuous cell lines can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256: 495-497, 1975), the trioma technique, the human B cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983) and the EBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to the polypeptides disclosed herein, or fragments comprising at least 5, 10, 15, 20, 25 , 30, 35, 40, 50, 75, 100 or 150 consecutive amino acids thereof. Alternatively, transgenic mice can be used to express humanized antibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides disclosed herein, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, can be used to detect systematically polypeptides from other organisms and samples. In these techniques, the polypeptides of the body are contacted with the antibody and those polypeptides that specifically bind to the antibody are detected. Any of the procedures described above can be used to detect binding to antibodies. One such screening test is described in Shulman H, Eberhard A, Eberhard C, Ulitzur S, Keinan E, Bioorg Med Chem. Lett. 2000 Oct. 16; 10 (20): 2353-6, Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria.

Kits

There are also kits comprising the compositions, such as nucleic acids, expression cassettes, vectors, cells, seeds or transgenic plants or plant parts, polypeptides (such as an aldolase enzyme) and / or antibodies disclosed herein. The kits also contain instructional material that teaches the industrial, medical, and dietary uses and methodologies disclosed herein, as described herein.

Whole cell manipulation and measurement metabolic parameters

The methods disclosed herein provide for the evolution of whole cells, or the manipulation of whole cells, of a cell to develop a new strain having a new phenotype, such as new or modified aldolase activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase, by modifying the genetic makeup of the cell. See United States Patent Application No. 20040033975.

The genetic makeup can be modified by adding to the cell a nucleic acid disclosed herein, such as a coding sequence for an enzyme disclosed herein. See WO0229032 and WO0196551.

To detect a new phenotype, at least one metabolic parameter of a modified cell is monitored in the cell in "real time" or "online". In some embodiments, a plurality of cells, such as a cell culture, are monitored in "real time" or "online". In some embodiments, a plurality of metabolic parameters are monitored in "real time" or "online". Metabolic parameters can be monitored using the aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein.

Metabolic Flow Analysis (MFA) is based on a known biochemical framework. A linearly independent metabolic matrix is constructed based on the law of conservation of mass and on the hypothesis of the pseudo-stationary state (PSSH) in intracellular metabolites. In the implementation of the methods disclosed herein, metabolic networks are established, including:

-

 identity of all substrates, products and intermediate metabolites of the pathways

-

 identity of all chemical reactions that interconvert pathway metabolites, stoichiometry of pathway reactions,

-

 the identity of all the enzymes that catalyze the reactions, the kinetics of the enzymatic reactions,

-

 regulatory interactions between pathway components, such as allosteric interactions, enzyme-enzyme interactions, etc.,

-

 intracellular compartmentalization of enzymes or any other supramolecular organization of enzymes, and

-

 the presence of any concentration gradient of metabolites, enzymes or effector molecules or diffusion barriers of its movement.

Once the metabolic network for a particular strain has been constructed, the mathematical presentation can be introduced using the notion of matrix to estimate intracellular metabolic fluxes if online metabolomic data is available. The metabolic phenotype depends on changes in the entire metabolic network within a cell. The metabolic phenotype depends on changes in the use of the pathways with respect to environmental conditions, genetic regulation, state of development of the genotype, etc. In some embodiments of the methods disclosed herein, after calculating the MFA online, the dynamic behavior of the cells, their phenotype, and other properties are analyzed by investigating the use of the pathways. For example, if glucose supply increases and oxygen decreases during yeast fermentation, airway utilization will decrease and / or stop and dominate fermentation pathway utilization. Control of the physiological status of cell cultures will be possible after analysis of the pathways. The methods disclosed herein can help determine how to manipulate fermentation by determining how to change supply, temperature, use of inductors, etc. of the substrate to monitor the physiological state of the cells to move in the desirable direction. In implementing the methods disclosed herein, the MFA results can also be compared with the transcriptome and proteome data to design experiments and protocols for metabolic manipulation or gene transposition, etc.

In practicing the methods disclosed herein, any new or modified phenotype can be conferred and detected, including new or improved characteristics in the cell. Any aspect of metabolism or growth can be monitored.

Monitoring the expression of an mRNA transcript

In some embodiments, the manipulated phenotype comprises increasing or decreasing the expression of an mRNA transcript (such as an aldolase enzyme message, such as pyruvate aldolase, HMG and / or KHG aldolase) or generating new transcripts (such as aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase) in a cell. This increased or decreased expression can be traced by tests for the presence of an aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase according to the invention, or by assays for aldolase enzyme activity, such as pyruvate aldolase, such as HMG and / or KHG aldolase. MRNA transcripts, or messages, can also be detected and quantified by any method known in the art, such as Northern blotting, quantitative amplification reactions, hybridization with arrays, and the like. Quantitative amplification reactions include, for example, quantitative PCR, including, for example, the polymerase chain reaction with quantitative reverse transcription or RT-PCR; Quantitative real-time RT-PCR or "real-time kinetic RT-PCR" (see Kreuzer (2001) Br. J. Haematol. 114: 313-318; Xia (2001) Transplantation 72: 907-914).

In some embodiments, the manipulated phenotype is generated by disabling the expression of a homologous gene. The coding sequence or one or more transcriptional control elements of the gene, such as promoters or enhancers, can be turned off. In this way, the expression of a transcript can be completely eliminated or only reduced.

In some embodiments, the manipulated phenotype comprises increasing the expression of a homologous gene. This can be accomplished by deactivating a negative control element, including a cis- or trans-acting transcriptional regulatory element, or by mutagenizing a positive control element. One or more or all of the transcripts in a cell can be measured by hybridization of a sample comprising cell transcripts or nucleic acids that represent or complement transcripts of a cell, by hybridization of immobilized nucleic acids in an array.

Monitoring the expression of polypeptides, peptides and amino acids

In some embodiments, the manipulated phenotype comprises increasing or decreasing the expression of a polypeptide (such as an aldolase enzyme, such as pyruvate aldolase, HMG and / or KHG aldolase) or generating new polypeptides in a cell. This increased or decreased expression can be traced by determining the amount of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase present, or by assays for activity of the aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase. Polypeptides, peptides and amino acids can also be detected and quantified by any method known in the art, including, for example, nuclear magnetic resonance (NMR), spectrophotometry, radiography (radiolabeling of proteins), electrophoresis, capillary electrophoresis, high performance liquid chromatography ( HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, different immunological methods, such as immunoprecipitation, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), immuno-fluorescent assays, electrophoresis in gel (such as SDS-PAGE), antibody staining, fluorescence activated cell sorter (FACS), pyrolysis mass spectrometry, Fourier transform infrared spectrometry, Raman spectrometry, GC-MS and LC-electrospray, and mass spectrometry by tandem cap-LC electrospray and the like. Novel bioactivities can also be systematically detected using methods, or variations thereof, described in US Patent No.

6,057,103. Furthermore, as detailed below, one, more than one or all of the polypeptides in a cell can be measured using a protein array.

Industrial, pharmaceutical and other applications

The polypeptides disclosed herein (such as those with aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase) can catalyze the formation or cleavage of carbon-carbon bonds. The enzymes disclosed herein can be highly selective catalysts. There are industrial processes that use enzymes disclosed herein, such as in the pharmaceutical or nutritional (dietary) supplement industry and in the food and feed industry, such as in methods for making food and feed products or additives for food and feed. There are processes that use enzymes disclosed herein in the medical industry, such as for the manufacture of pharmaceutical or auxiliary products or dietary supplements or food supplements and additives.

Biomass conversion and clean biofuel production

There are enzymes, such as aldolases, including pyruvate aldolases such as, but not limited to, HMG and / or KHG aldolases (including mixtures or "cocktails" of enzymes) and methods for the conversion of a biomass or any lignocellulosic material (eg, any composition comprising cellulose, hemicellulose, and lignin) in fuels (eg, bioethanol, biobutanol, biopropanol, biomethanol, biodiesel), using the enzymes disclosed herein, in addition to animal and human food and chemicals. Thus, the compositions and methods disclosed herein are sustainable alternatives or adjuvants for use as petroleum-based products, for example, a mixture of bioethanol and gasoline. There are organisms that express enzymes disclosed herein for participation in chemical cycles involving the natural conversion of biomass. In one embodiment, enzymes and methods for conversion are used in sets of enzymes for efficient depolymerization of cellulosic and hemicellulosic polymers and obtaining metabolizable carbon residues. There are also methods to discover and implement the most efficient enzymes to enable these important new industrial processes of "biomass conversion" and alternative energy.

The methods disclosed herein also include taking the converted lignocellulosic material (processed by enzymes of the invention) and converting it into fuel (eg, bioethanol, biobutanol, biopropanol, biomethanol, biodiesel) by fermentation and / or chemical synthesis. In one embodiment, the sugars produced are fermented and / or the non-fermentable products are gasified.

The enzymes disclosed herein (including, for example, organisms, such as microorganisms, e.g., fungi, yeast, or bacteria, that generate and in some embodiments secrete recombinant enzymes disclosed herein) can be used or included / integrated at any stage from any biomass conversion process, for example, in any step, in several steps or included in all steps or in all the following methods of biomass conversion processes or all these biofuel alternatives:

Direct combustion: burning of material by direct heat and is the simplest biomass technology; It can be very economical if there is a biomass source nearby.

Pyrolysis: is the thermal degradation of biomass by heat in the absence of oxygen. In one embodiment, the biomass is heated to a temperature between about 800 and 1,400 degrees Fahrenheit but no oxygen is introduced to support combustion resulting in the creation of gas, fuel oil, and coal.

Gasification: biomass can be used to produce methane through heating or anaerobic digestion. Synthesis gas, a mixture of carbon monoxide and hydrogen, can be derived from biomass.

Landfill gas: is generated by the decomposition (anaerobic digestion) of garbage buried in landfills. When organic waste decomposes, it generates gas consisting of approximately 50% methane, the main component of natural gas.

Anaerobic digestion: converts organic matter into a mixture of methane, the main component of natural gas, and carbon dioxide. In one embodiment, biomass such as wastewater (wastewater), manure, or food processing waste is mixed with water and fed to an airless digester tank.

Fermentation

Alcohol Fermentation: Fuel alcohol is produced by converting starch into sugar, fermenting the sugar to obtain alcohol, and then separating the mixture of alcohol and water by distillation. Raw materials such as wheat, barley, potato, and paper waste, sawdust, and straw, which contain sugar, starch, or cellulose, can be converted to alcohol by starch fermentation.

Transesterification: An exemplary reaction to convert oil to biodiesel is called transesterification. In the transesterification process, an alcohol (such as methanol) is reacted with triglyceride oils found in vegetable oils, animal fats, or recycled fats, forming alkyl esters of fatty acids (biodiesel) and glycerin. The reaction requires heat and a strong base catalyst, such as sodium hydroxide or potassium hydroxide.

Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters made from recycled vegetable oils, animal fats, or fats. Biodiesel can be used as a vehicle fuel in its pure form, but is normally used as an additive for petroleum diesel to reduce the levels of particulates, carbon monoxide, hydrocarbons, and toxic air products from vehicles with diesel engines.

Hydrolysis: Includes hydrolysis of a compound, eg, biomass, such as a lignocellulosic material, catalyzed using an enzyme disclosed herein.

Cogeneration: is the simultaneous production of more than one form of energy using a single fuel and the same facilities. In one embodiment, biomass cogeneration has more potential growth than biomass generation alone because the cogeneration produces heat and electricity.

In one embodiment, the polypeptides disclosed herein have aldolase activity, including pyruvate aldolase activity, such as, but not limited to, HMG and / or KHG aldolase activity, or other enzymatic activity to generate biodiesel, bioethanol, biobutanol , biopropanol or biomethanol from an organic material, for example, a biomass, such as compositions derived from plants and animals, including any agricultural crop or other renewable raw material, agricultural residue or animal waste, or organic components of waste municipal and industrial, or microorganisms such as algae or yeast. In one embodiment, the polypeptides disclosed herein are used in processes to convert lignocellulosic biomass to ethanol, butanol, propanol, methanol, or are otherwise used in processes to hydrolyze or digest biomaterials such that they can be used as a biofuel (including bioethanol, biobutanol, biopropanol, biomethanol or biodiesel), or to simplify the processing of biomass to obtain a fuel. In an alternative embodiment, the polypeptides disclosed herein are used in processes for a transesterification process by reacting an alcohol (such as methanol) with a triglyceride oil in a vegetable oil, animal fat, or recycled fats, forming alkyl esters of acids fatty (biodiesel) and glycerin. In one embodiment, the biodiesel is obtained from recycled soybean oil or cooking oils. Animal fats, other vegetable oils, and other recycled oils can also be used to produce biodiesel, depending on cost and availability. In other embodiments, mixtures of all types of fats and oils are used to produce biodiesel fuel of the invention.

Enzymes disclosed herein can also be used in glycerin refining. The glycerin-derived product contains unreacted catalyst and soaps that are neutralized with an acid. Water and alcohol are removed to produce 50% to 80% crude glycerin. The remaining contaminants include unreacted fats and oils, which can be processed using the polypeptides disclosed herein. In large biodiesel plants disclosed herein, glycerin can be further purified, for example up to 99% or more purity, for the pharmaceutical and cosmetic industries.

The bioethanol, biobutanol, biopropanol, biomethanol and / or biodiesel obtained using the polypeptides disclosed herein can be used with fuel oxygenators to improve combustion characteristics. Adding oxygen results in more complete combustion, which reduces carbon monoxide emissions. This is another environmental benefit of replacing petroleum fuels with biofuels (eg, a fuel of the invention). A bioethanol, biobutanol, biopropanol, biomethanol, and / or biodiesel made using the compositions and / or methods disclosed herein can be mixed with gasoline to form an E10 blend (approximately 5% to 10% ethanol and approximately 90% to 95% of gasoline), but can be used in higher concentrations, such as E85 or in its pure form. A bioethanol, biobutanol, biopropanol, biomethanol and / or biodiesel obtained using the compositions and / or methods disclosed herein can be mixed with petroleum diesel to form a B20 mixture (20% biodiesel and 80% petroleum diesel), although Other mix levels up to B100 (pure biodiesel) can be used.

There are also processes for making ethanol ("bioethanol"), butanol ("biobutanol"), propanol ("biopropanol"), methanol ("biomethanol") and / or diesel ("biodiesel") from compositions comprising lignocellulosic biomass. Lignocellulose biomass material can be obtained from agricultural crops, as a product derived from the production of food for humans and animals, or as lignocellulosic waste products, such as plant residues and waste paper. Examples of plant sources or plant residues suitable for treatment with polypeptides disclosed herein include kelp, algae, grains, seeds, stems, leaves, pods, husks, corn cobs, corn residues, straw, grasses (eg grass Indian, such as Sorghastrum nutans; or rod grass, for example, the species Panicum, such as Panicum virgatum) and the like, as well as wood, wood chips, wood pulp, and sawdust. Examples of paper wastes suitable for treatment with polypeptides disclosed herein include discarded photocopy paper, computer printer paper, notebook paper, notebook paper, typewriter paper, and the like, as well as newspapers, magazines, cardboard and paper-based packaging materials.

In one embodiment, the enzymes and methods disclosed herein can be used in conjunction with more "traditional" means to obtain ethanol, methanol, butanol, propanol and / or diesel from biomass, for example, as methods comprising hydrolyzing lignocellulosic materials by subjecting dry lignocellulosic material in a reactor to a catalyst consisting of a dilute solution of a strong acid and a metal salt; this can reduce the activation energy, or the temperature, of cellulose hydrolysis to obtain higher sugar yields; see, for example, United States Patent Nos. 6,660,506; and 6,423,145.

Another embodiment incorporating the use of enzymes disclosed herein comprises hydrolyzing hemicellulose, cellulose and lignin containing lignocellulosic material by subjecting the material to a first stage hydrolysis step in an aqueous medium at a selected temperature and pressure in order to produce mainly the depolymerization of hemicellulose without a significant depolymerization in the conversion of cellulose to glucose. This step results in a suspension in which the liquid aqueous phase contains dissolved monosaccharides resulting from the depolymerization of hemicellulose and a solid phase containing cellulose and lignin. A second stage hydrolysis step may comprise conditions such that at least a significant portion of the cellulose is depolymerized, said step resulting in a liquid aqueous phase containing the dissolved / soluble cellulose depolymerization products. See, for example, United States Patent No. 5,536,325. The enzymes disclosed herein can be added at any stage of this exemplary process.

Another embodiment incorporating the use of enzymes disclosed herein comprises processing a lignocellulose-containing biomass material by one or more acid hydrolysis steps diluted with about 0.4% to 2% strong acid; and treating an unreacted solid lignocellulosic component of the acid hydrolyzed biomass material by alkaline delignification to produce precursors for thermoplastics and biodegradable derivatives. See, for example, United States Patent No. 6,409,841. The enzymes disclosed herein can be added at any stage of this exemplary process.

Another embodiment incorporating the use of enzymes disclosed herein comprises prehydrolyzing lignocellulosic material in a prehydrolysis reactor; adding an acidic liquid to the solid lignocellulosic material to prepare a mixture; heat the mixture to reaction temperature; maintaining the reaction temperature for a time sufficient to fractionate the lignocellulosic material and obtain a solubilized portion containing at least about 20% of the lignin of the lignocellulosic material and a solid fraction containing cellulose; removing a solubilized portion of the solid fraction while it is at the reaction temperature or a temperature close to where the cellulose in the solid fraction becomes more suitable for enzymatic digestion; and recovering a solubilized portion. See, for example, United States Patent No. 5,705,369. The enzymes disclosed herein can be added at any stage of this exemplary process.

The invention provides methods for obtaining engine fuel compositions (eg, for spark ignition engines) based on liquid hydrocarbons mixed with a fuel grade alcohol obtained using an enzyme or a method disclosed herein. In one embodiment, fuels obtained by using an enzyme disclosed herein comprise, for example, mixtures of carbon gas liquid or natural gas liquid-ethanol, methanol, butanol, propanol and / or diesel. In one embodiment, a co-solvent is biomass-derived 2-methyltetrahydrofuran (MTHF). See, for example, United States Patent No. 6,712,866.

In one embodiment, the methods of the invention for the enzymatic degradation of lignocellulose, for example, for the production of ethanol from lignocellulosic material, can also comprise the use of ultrasonic treatment of the biomass material; see, for example, United States Patent No. 6,333,181.

In another embodiment, the methods disclosed herein for producing bioethanol, biobutanol, biopropanol, biomethanol, and / or biodiesel from a cellulosic substrate comprise providing a reaction mixture in the form of a suspension comprising a cellulosic substrate, an enzyme disclosed in it and a fermentation agent (eg, within a reaction vessel, such as a semi-continuous solids feed bioreactor), and the reaction mixture is reacted under conditions sufficient to initiate and maintain a fermentation reaction ( as described, for example, in United States Patent Application No. 20060014260). In one embodiment, experimental or theoretical calculations can determine an optimal feed frequency. In one embodiment, additional amounts of the cellulosic substrate and enzyme are provided to the reaction vessel at intervals according to the optimized feed frequency.

An exemplary process for the manufacture of biofuels (such as bioethanol, biobutanol, biopropanol, biomethanol, and / or biodiesel) is described in United States Patent Applications Published Nos. 20050069998; 20020164730; and in one embodiment it comprises the steps of grinding lignocellulosic biomass (for example, up to a size of 15-30 mm), subjecting the obtained product to a steam blast pre-treatment (for example, at a temperature of 190-230ºC). ) for between 1 and 10 minutes in a reactor; collecting pre-treated material in a cyclone or related manufacturing product; and separating the liquid and solid fractions by filtration on a filter press, introducing the solid fraction into a fermentation tank and adding one or more enzymes disclosed herein, eg, a cellulase and / or beta-glucosidase (eg, dissolved in citrate buffer solution pH 4.8).

Another exemplary process for making biofuels (such as bioethanol, biobutanol, biopropanol, biomethanol, and / or biodiesel) comprising using enzymes disclosed herein involves pre-treating a starting material comprising a lignocellulosic raw material comprising at least hemicellulose and cellulose . In one embodiment, the starting material comprises potatoes, soybeans (rapeseed), barley, rye, corn, oats, wheat, beets or sugar beets or a component or waste or product derived from food or feed production. The starting material ("raw material") is reacted under conditions that alter the structure of the plant fiber to produce at least a partial hydrolysis of hemicellulose and cellulose. The alteration conditions may comprise, for example, subjecting the starting material to an average temperature of 180 ° C to 270 ° C at a pH of 0.5 to 2.5 over a period of from about 5 seconds to 60 minutes; or a temperature of 220 ° C to 270 ° C at a pH of 0.5 to 2.5 over a period of 5 seconds to 120 seconds or equivalent. This generates a raw material with greater accessibility for its digestion by an enzyme, for example, a cellulase enzyme of the invention. United States Patent No. 6,090,595.

Exemplary conditions for the hydrolysis of lignocellulosic material include reactions at temperatures between about 30 ° C and 48 ° C and / or a pH between about 4.0 and 6.0. Other exemplary conditions include a temperature between about 30 ° C and 60 ° C and a pH between about 4.0 and 8.0.

The enzymes disclosed herein can catalyze reactions with high stereo-, regio-, and chemo-selectivity. The aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein can be manipulated to operate in different solvents, operate at extreme pHs (eg, high pHs and low pHs), extreme temperatures (eg, high temperatures and low temperatures), extreme salinity levels (eg, high salinity and low salinity) and catalyze reactions with compounds that are not structurally related to their natural physiological substrates.

Feed and food or feed and food additives

In addition to providing dietary aids or supplements, or food supplements and additives, there are compositions and methods for treating human and animal feed and food and feed and feed additives using a polypeptide disclosed herein, such as a protein having aldolase enzyme activity. , such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, and / or the antibodies disclosed herein. There are animal feeds, food and additives comprising aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein and / or antibodies disclosed herein. The animal can be any farm animal or any animal.

The animal feed additive disclosed herein can be a granulated enzyme product that can be easily mixed with the feed components. Alternatively, the feed additives disclosed herein can form a component of a premix. The granulated enzyme product disclosed herein can be coated or uncoated. The particle size of the enzyme granules may be compatible with that of the feed and pre-mix components. This provides a safe and convenient means of incorporating enzymes into feed. Alternatively, the animal feed additive disclosed herein can be a stabilized liquid composition. This can be an aqueous or oily base suspension. See United States Patent No. 6,245,546.

The aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, in modifying feed or food, can process the food or feed in vitro (by modifying the components of the feed or food) or in alive. The polypeptides disclosed herein can be added to feed or food compositions.

In some embodiments, an enzyme disclosed herein is added in combination with another enzyme, such as beta-galactosidases, catalases, laccases, cellulases, other aldolases, endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, glucosidases, glucose isomerases. , glycosyltransferases, lipases, phospholipases, lipoxygenases, beta-laccases, endo-beta-1,3 (4) -laccases, cutinases, peroxidases, amylases, phytases, glucoamylases, pectinases, reductases, oxidases, decarboxylases, lignins, lignins, arabininases, hemicellulases, mannanases, xylolacasses, xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases, polygalacturonases, rhamnogalacturinases, pectin liases, transglutainase, lactosetase, and transglutaminase, digestible by the animal. In this way, the aldolase enzymes, such as pyruvate aldolase, such as HMG and / or KHG aldolase disclosed herein, can contribute to the available energy of the feed or food or to the digestibility of the food or feed by decomposing the cellulose.

In other embodiments, the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein can be supplied by expressing the enzymes directly in transgenic feed crops (such as transgenic plants and seeds and the like), such as grains, cereals, corn, soybeans, rapeseed, lupine and the like. As stated above, there are transgenic plants, plant parts, and plant cells that comprise a nucleic acid sequence encoding a polypeptide disclosed herein. In some embodiments, the nucleic acid is expressed such that the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase, disclosed herein is produced in recoverable amounts. The aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase can be recovered from any plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such to improve the quality of a food or feed, such as improving nutritional value, palatability, etc.

In some embodiments, the enzyme delivery matrix disclosed herein is in the form of discrete plural particles, granules, or granules. "Granules" means particles that are compressed or compacted, for example by granulation, extrusion, or the like, to remove water from the matrix. Such compression or compaction of the particles also promotes interparticle cohesion of the particles. For example, granules can be prepared by granulating the grain-based substrate in a granulate mill. The granules prepared in this way are milled or minced to a granule size suitable for use as an adjuvant in animal feed. Since it is approved for use in animal feed, the matrix can be used as a diluent for the administration of enzymes in animal feed.

In some embodiments, the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase contained in the enzyme delivery matrix and in the methods is a thermostable aldolase enzyme, such as pyruvate aldolase, such as HMG and / or KHG aldolase, as described herein, in order to resist inactivation of the enzyme aldolase, such as pyruvate aldolase, such as HMG and / or KHG aldolase during manufacture, in which elevated temperatures and / or steam may be employed to prepare the granulated enzyme delivery matrix. During digestion of feed containing the enzyme delivery matrix, aqueous digestive fluids will cause the release of the active enzyme. Other types of thermostable enzymes and nutritional supplements that are thermostable can also be incorporated into the delivery matrix to be released under any type of aqueous conditions.

In some embodiments, a coating is applied to the enzyme matrix particles for many different purposes, such as adding a flavor or nutritional supplement to the animal feed, delaying the release of supplements and enzymes into the animal feed under gastric and similar conditions. In some embodiments, the coating is applied to achieve a functional objective, for example, when it is desirable to slow down the release of the enzyme from the matrix particles or to control the conditions under which the enzyme will be released. The composition of the coating material may be such that it is selectively degraded by the agent to which it is susceptible (such as heat, acid or base, enzymes, or other chemical). Alternatively, two or more coatings susceptible to said different degradation agents can be applied consecutively to the matrix particles.

There is also a process to prepare an enzyme release matrix. The process comprises providing discrete plural particles of a grain-based substrate in a particle size suitable for use as an enzyme-releasing matrix, wherein the particles comprise an enzyme aldolase, such as pyruvate aldolase, HMG and / or KHG aldolase. encoded by an amino acid sequence disclosed herein. In some embodiments, the process includes compacting or compressing the enzyme-releasing matrix particles to form granules, which is largely accomplished by granulation. The mold and cohesive inhibiting agent, if used, can be added at any suitable time and, in some embodiments, mixed with the grain-based substrate in the desired proportions prior to granulation of the grain-based substrate. The moisture content in the granulate mill in some embodiments is in the ranges set forth above with respect to the moisture content in the finished product and, in some embodiments, is approximately 14-15%. In some embodiments, moisture is added to the raw material in the form of an aqueous enzyme preparation to adjust the raw material to this moisture content. The temperature in the granulate mill in some embodiments is brought up to about 82 ° C with steam. The granulate mill can be operated in any condition that imparts sufficient work to the raw material to provide granules. The granulation process itself is an inexpensive process to remove water from the enzyme-containing composition.

The compositions and methods disclosed herein can be practiced in conjunction with the administration of prebiotics, which are high molecular weight sugars, such as fructo-oligosaccharides (FOS); galacto-oligosaccharides (GOS), GRAS material (generally recognized as safe). These prebiotics can be metabolized by some probiotic lactic acid bacteria (LAB). They are not digestible by most gut microbes.

Food processing and processing

There are foods and feeds that comprise enzymes disclosed herein and methods of using enzymes disclosed herein in food and feed processing. The aldolase enzymes, such as pyruvate aldolase, HMG and / or KHG aldolases disclosed herein have numerous applications in the food processing industry. There are methods of hydrolyzing compositions comprising cellulose including, for example, a plant cell, a bacterial cell, a yeast cell, an insect cell or an animal or any plant or part of a plant or any food or feed or a product of waste and the like.

For example, there are foods or feeds that comprise an aldolase enzyme, such as pyruvate aldolase, HMG, and / or KHG aldolase, disclosed herein, such as in a feed, a liquid, such as a beverage (such as fruit juice or a beer), a bread or a dough or a baked product or a precursor of a drink (such as a beer or must).

The food treatment processes disclosed herein may also include the use of any combination of other enzymes such as tryptophanases or tyrosine decarboxylases, laccases, catalases, laccases, other aldolases, cellulases, endoglycosidases, endo-beta-1,4-laccases. , amyloglucosidases, glucosidases, glucose isomerases, glycosyltransferases, lipases, phospholipases, lipoxygenases, beta-laccases, endo-beta-1,3 (4) -laccases, cutinases, peroxidases, amylases, phytases, glucoamylases, pectinases, reductases, , phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xylolacases, xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, proteases, peptidases, transinases, polygalacturonases, rhamnogalacturonases, galactanases, galactanases

Pharmaceutical compositions and dietary supplements

There are pharmaceutical compositions and dietary supplements (such as dietary aids) that comprise an aldolase disclosed herein. Aldolase activity comprises pyruvate aldolase, HMG and / or KHG aldolase activity. In some embodiments, the pharmaceutical compositions and dietary supplements (such as dietary aids) are formulated for oral ingestion.

Periodontal treatment compounds may comprise an enzyme disclosed herein, such as described in US Patent No. 6,776,979. Compositions and methods for the treatment or prophylaxis of acid bowel syndrome can comprise an enzyme disclosed herein, as described in US Patent No. 6,468,964.

In other embodiments, wound dressings, implants, and the like comprise antimicrobial enzymes (such as antibiotics), including an enzyme disclosed herein (including, for example, sequences disclosed herein). The enzymes disclosed herein can also be used in alginate dressings, antimicrobial barrier dressings, burn dressings, compression bandages, diagnostic tools, gel dressings, hydrocellular (foam) dressings, hydrocolloid dressings, IV dressings, fields Incision, low-adhesion dressings, odor absorbent dressings, paste bandages, post-operative dressings, scar management, skin care, transparent film dressings and / or wound healing. The enzymes disclosed herein can be used in wound cleansing, in wound bed preparation, to treat pressure ulcers, leg ulcers, burns, diabetic foot ulcers, scars, IV fixation, surgical wounds, and minor wounds . The enzymes disclosed herein can be used in sterile enzyme debridement compositions, such as ointments. In various embodiments, aldolase is formulated as a tablet, gel, pill, implant, liquid, aerosol, film, micelle, powder, food, feed granulate, or as an encapsulated formulation.

The pharmaceutical compositions and dietary supplements disclosed herein may also include the use of any combination of other enzymes, such as beta-galactosidases, catalases, laccases, cellulases, other aldolases, endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, glucosidases, glucose isomerases, glycosyltransferases, lipases, phospholipases, lipoxygenases, beta-laccases, endo-beta-1,3 (4) -laccases, cutinases, peroxidases, amylases, phytases, glucoamylases, pectinases, reductases, Phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xylolarases, xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, proteases, peptidases, translutains, translutains, galactanases, galactanases, galactanases

Biosynthetic pathways to produce R, R and other monatin stereoisomers

As described, inter alia, in WO 03/091396 A2 (see Figures 1-3 and 11-13), monatin can be produced from tryptophan via a multi-stage pathway involving biological conversions ( that is, it facilitates the reaction of a substrate to form a product with a polypeptide). One disclosed route involves biologically converting tryptophan to indole-3-pyruvate, biologically converting indole-3-pyruvate to 2-hydroxy 2- (indole-3-ylmethyl) -4-keto glutaric acid ("MP"), and biologically converting the MP in monatina. In some embodiments, the polypeptides disclosed herein can be used to facilitate the reaction of indole-3-pyruvate to form MP. In some embodiments, the polypeptides disclosed herein can be used to preferably facilitate the production of R-MP.

In some embodiments, one or more polypeptides selected from isolated or recombinant polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO : 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 , SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO : 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 , SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ED NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, S EQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 2 60, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof that have aldolase activity may be useful in facilitating a reaction within a multistage pathway to producing a selected product of monatin, monatin derivatives, salts thereof, and combinations thereof as further defined in the claims. In one embodiment, aldolase-active polypeptides may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives , salts thereof and combinations thereof.

In another embodiment, one or more polypeptides selected from isolated or recombinant polypeptides with HMG aldolase activity of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, I KNOW Q ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304 or Fragments or subsequences thereof that have aldolase activity may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a multi-step pathway to produce a selected monatin product derived from monatin, salts thereof, and combinations thereof, as further defined in the claims. In one embodiment, polypeptides with HMG aldolase activity may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product derived from monatin, salts thereof, and combinations thereof.

In yet another embodiment, one or more polypeptides selected from isolated or recombinant polypeptides with KHG aldolase activity of any of SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO : 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330 , SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof having aldolase activity may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a pathway of multiple steps to produce a selected monatin product, monatin derivatives, salts thereof, and combinations thereof, as further defined in the claims. In one embodiment, polypeptides with KHG aldolase activity may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, derivatives monatin, salts thereof and combinations thereof.

Additionally, one or more polypeptides encoded by one or more nucleic acid sequences having at least about 95%, 96%, 97%, 98%, 99% or more, or full sequence identity (100%) with an acid nucleic acid disclosed herein, including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO : 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 , SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO : 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97 , SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID N O: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, I KNOW THAT ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 253, SEQ ID NO : 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271 , SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 303, SEQ ID NO : 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321 , SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives , salts thereof or combinations thereof as further defined in the claims. In one embodiment, the polypeptide (s), or fragments or subsequences thereof with aldolase activity may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to producing a selected product of monatin, monatin derivatives, salts thereof, and combinations thereof as further defined in the claims.

One or more polypeptides with HMG aldolase activity encoded by a nucleic acid sequence having at least about 95%, 96%, 97%, 98%, 99% or more, or full sequence identity (100%) with a nucleic acid according to the invention, including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO : 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 , SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO : 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a multi-step pathway to produce a selected product of monatin, monatin derivatives, salts of the the same or combinations thereof as defined in the claims. In one embodiment, the HMG aldolase-active polypeptide or polypeptides may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as defined in the claims.

One or more polypeptides with KHG aldolase activity encoded by a nucleic acid sequence having at least about 95%, 96%, 97%, 98%, 99% or more, or full sequence identity (100%) with a nucleic acid as defined in the claims, including SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO : 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335 , SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a multi-step pathway to produce a product selected from monatin, monatin derivatives, salts thereof, and combinations thereof as further defined in the claims. In one embodiment, the polypeptide or polypeptides with KHG aldolase activity may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as further defined in the claims.

Likewise, one or more polypeptides with aldolase activity encoded by a nucleic acid sequence hybridizes under stringent conditions with a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 11 7, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ IDNO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a cap within a pathway. multiple steps to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as defined in the claims. In one embodiment, the aldolase-active polypeptide (s) may be useful to facilitate a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, derivatives monatin, salts thereof and combinations thereof as defined in the claims.

One or more polypeptides with HMG aldolase activity encoded by a nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO : 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 , SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO : 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 , SEQ ID NO: 109, SEQ ID NO: 11, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO : 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151 , SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO : 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201 , SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO : 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251 , SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO : 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 , SEQ ID NO: 303, SEQ ID NO: 305, may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as defined in the claims. In one embodiment, the HMG aldolase-active polypeptide or polypeptides may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as defined in the claims.

One or more polypeptides with KHG aldolase activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid of SEQ ID NO: 307, SEQ ID NO: 301, SEQ ID NO: 313 , SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, and SEQ ID NO: 338 can be helpful in facilitating a reaction between indole-3-pyruvate and a source C3 carbon as a step within a multi-step pathway to produce a selected product of monatin, monatin derivatives, salts thereof, and combinations thereof as further defined in the claims. In one embodiment, the polypeptide or polypeptides with KHG aldolase activity may be useful in facilitating a reaction in which indole-3-pyruvate is converted to MP as a step within a multi-step pathway to produce a selected monatin product, monatin derivatives, salts thereof and combinations thereof as further defined in the claims.

The aldolase-active polypeptides described herein may be useful in facilitating a reaction between indole-3-pyruvate and a C3 carbon source. The C3 carbon source may be, non-exhaustively, oxaloacetate, pyruvate or a pyruvate derivative, such as phosphoenolpyruvate. In one embodiment, the C3 carbon source is pyruvate.

Examples of enzymes useful for the conversion of the reaction product between indole-3-pyruvate and the C3 carbon source to monatin include members of the enzyme classes: tryptophan aminotransferases (2.6.1.27), tryptophan dehydrogenases (1.4.1.19), D -amino acid dehydrogenases (1.4.99.1), glutamate dehydrogenases (1.4.1.2-4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminases (2.6.1.28), or more generally members of the aminotransferase family (2.6 .1.-) such as aspartate aminotransferase (EC 2.6.1.1), tyrosine aminotransferase (aromatic) (2.6.1.5), D-tryptophan aminotransferase or D-alanine aminotransferase (2.6.1.21) (see Figure 2 of document WO 03 / 091396 A2). This reaction can also be carried out using chemical reactions. The amination of the keto acid (MP) is carried out by reductive amination using ammonia and sodium cyanoborohydide. Figures 11-13 of WO 03/091396 A2 show additional polypeptides that can be used to convert MP to monatin, as well as to provide higher yields of monatin from indole-3-pyruvate or tryptophan. In one embodiment, these enzymes are used to catalyze the conversion of MP, the reaction product between indole-3-pyruvate and pyruvate, to monatin.

The flavor profile of a monatin composition can be altered by controlling the relative amount of the various monatin stereoisomers in the composition. The present invention provides routes and substances to produce monatin compositions with a desired percentage of Monatin R, R and / or (S, R) -monatin.

The chirality of monatin compounds produced by the disclosed pathways can be affected by both pH and the polypeptides used for biological conversions. The aldolase-active polypeptides described herein can be used to control monatin carbon-2 chirality (see Formula I above) in the reaction in which indole-3-pyruvate is converted to MP.

Once the reaction product of the reaction between indole-3-pyruvate and the C3 carbon source is produced, the stereospecific amino group can be added. The R or S configuration of carbon-4 (see Formula I above) can be generated depending on whether aromatic acid D or L aminotransferase is used. Many aminotransferases are specific for the L-isomer; however, D-tryptophan aminotransferases exist in certain plants (Kohiba and Mito, Proceedings of the 8th International Symposium on Vitamin B6 and Carbonyl Catalysis, Osaka, Japan 1990). In addition, D-alanine aminotransferases (2.6.1.21), Dmethionine-pyruvate aminotransferases (2.6.1.41) and (R) -3-amino-2-methylpropanoate aminotansferases (2.6.1.61), (S) -3amino- have been identified. 2-methylpropanoate aminotransferase (2.6.1.22) and D-phenylglycine aminotransferase. Certain aminotransferases can accept only the substrate by this reaction with a particular configuration at the C2 carbon. Therefore, even if the conversion to the reaction product between indole-3-pyruvate and the carbon 3 source is not stereospecific, the stereochemistry of the final product can be controlled through the appropriate selection of aminotransferase. Since the reactions are reversible, the unreacted reaction product (unwanted isomer) can be recycled back to its constituents, and a racemic mixture of the reaction product can be re-formed.

An example of an amino donor suitable for addition of an amino group to a product of the reaction between indole-3-pyruvate and the C3 carbon source includes, but is not limited to, an amino acid, such as alanine, aspartate, lysine , glutamate, glycine and tryptophan.

With reference now to the figures, the following should be noted. Flow charts identify pathways to produce monatin, but are not limited to a particular method of implementing the pathways. For example, the pathways can be practiced in vivo, in vitro, or a combination thereof.

Furthermore, implementing the pathways does not require that each of the identified components (such as reagents and enzymes) be explicitly provided by the healthcare professional, to the extent that sufficient components, or sources of compounds, and conditions are provided. of reaction so that the route can potentially proceed. In other words, for example, if a figure represents a process for producing a monatin composition, which includes producing indole-3-pyruvate from L-tryptophan, producing 2-hydroxy 2- (indole-3-ylmethyl) acid - 4-keto glutaric ("monatin precursor" or "MP") from indole-3-pyruvate, and producing monatin from MP, where each reaction is facilitated by an appropriate enzyme, it is contemplated that Practice the pathway includes combining L-tryptophan with α-ketoglutarate and contemplated enzymes to facilitate the identified reactions, and under conditions suitable for each reaction to occur without explicitly also providing indole-3-pyruvate or MP. In that case, L-tryptophan could react with α-ketoglutarate to produce indole-3-pyruvate. Due to the conditions set and the enzyme provided, the indole-3-pyruvate produced from the L-tryptophan reaction could react to form MP, and then due to the established conditions and the enzyme provided, the MP produced from the indole-3-pyruvate reaction could react to form monatin.

It should also be noted that the pathways depicted do not require the health care provider to explicitly supply the identified starting materials or enzymes. In other words, it is contemplated that practicing any of the pathways that identify L-tryptophan as a starting material would include providing a compound that can produce L-tryptophan, under conditions suitable for L-tryptophan production to occur and combining such compound with enzymes capable of facilitating the series of reactions established under conditions that would be suitable for said reactions to occur. As another example, it is also contemplated that practicing the identified route would include providing a genetically engineered microorganism to produce monatin according to the described route, and providing appropriate conditions for the fermentation process to occur. For example, a microorganism, which naturally produces large amounts of L-tryptophan, could be genetically engineered to produce or overproduce one or more of the enzymes used to facilitate reactions on the monatin pathway, and appropriate conditions could be provided so that the microorganism produce monatin.

Figure 1 identifies the particular embodiment in which an R-specific aldolase facilitates the reaction of indole-3-pyruvate and pyruvate to form R-MP. The flow chart in Figure 1 schematically represents a process according to the invention for making a monatin composition including (R, R) -monatin. As shown in Figure 1, the general route involves a reaction of tryptophan to form indole-3-pyruvate to produce MP, and a reaction of MP to produce monatin, including (R, R) -monatin.

Figure 1 further illustrates specific permutations of this general pathway designed to increase the production of monatin form R, R at the expense of monatin forms S, S, R, S, and S, R. In particular, Figure 1 illustrates the embodiment where: the aminotransferase enzyme used in the L-tryptophan reaction has greater activity and / or specificity for said reaction with respect to the reactions of MP and monatin 4S or the oxidase has greater activity and / or specificity for L-tryptophan than for 4R monatin; the enzyme that facilitates the reaction of indole-3-pyruvate is a polypeptide with aldolase activity disclosed herein and the enzyme that facilitates the reaction of MP is a broad specificity D enzyme, preferably evolved to function more efficiently with the R isomer of MP.

Figure 1 also illustrates particular permutations designed to make the production of (R, R) monatin more economical. For example, in Figure 1, L-tryptophan — unlike D-tryptophan or combinations of L- and D-tryptophan — is identified as the starting material. While the choice of the specific form of tryptophan does not affect the chirality of the final monatin compounds in the monatin composition (because the tryptophan reaction forms indole-3-pyruvate, which has no chirality), some may prefer to use L- tryptophan as a starting material at least because L-tryptophan is currently less expensive and can be obtained more easily than D-tryptophan.

Focusing now on the first reaction shown in Figure 1, when tryptophan is converted to indole-3-pyruvate any one or more of alpha-ketoglutarate, oxaloacetate, and pyruvate reacts to form an amino acid (glutamate, aspartate, and alanine, respectively). Figure 1 depicts the embodiment where the tryptophan starting material is L-tryptophan, and alpha-ketoglutarate, oxaloacetate and / or pyruvate produce the L-isomeric form of the amino acid (such as L-glutamate, L-aspartate and / or L-Alanine, respectively).

As shown in Figure 1, an approach to improve the production of (R, R) -monatin derivatives involves facilitating the reaction of L-tryptophan with an enzyme that has higher specificity, higher activity, or both for tryptophan unlike MP or monatin, and facilitates the reaction of MP with a D enzyme. As disclosed in WO 03/091396 A2, certain enzymes can facilitate the reaction of tryptophan to produce indole-3-pyruvate, as well as the amination reaction of MP to produce monatina. The use of an L-aminotransferase in the amination stage creates a chiral center S at position C-4 of monatin, while the use of an enzyme D creates a chiral center at position C-4 of monatin. Therefore, in the case where L-aminotransferase, which facilitates the tryptophan reaction, is also active in the MP reaction, (R, S) - and (S, S) -monatin can be formed, depending on the MP form present. Furthermore, certain other enzymes — L-amino acid oxidases — may not only facilitate the reaction of tryptophan to form indole-3-pyruvate, but may have a secondary activity for the degradation of (R, R) -monatin. According to some embodiments, this secondary 4R activity is minimized or eliminated. Secondary oxidase activity in the 4S forms of monatin would reduce or minimize them in the final product and might be desirable depending on the desired final composition. Therefore, the greater the specificity and / or activity of the selected L enzyme for tryptophan with respect to MP or monatin, the greater the amount of R, R and S, R produced with respect to (S, S) - and (R, S) -monatin.

Suitable enzymes for the tryptophan reaction, according to the embodiment illustrated in Figure 1, include: Laminotransferases capable of facilitating a reaction of L-tryptophan to form indole-3-pyruvate, and having higher specificity for said reaction with respect to the reaction of R-MP to form 4S isomers of monatin; and, Lamino acid oxidases capable of facilitating a reaction of L-tryptophan to form indole-3-pyruvate, and which have greater specificity and / or activity for said reaction with respect to the reaction of 4R isomers of monatin to form MP, and functional equivalents. of any of the above. More specifically, non-exhaustive examples of suitable enzymes can be selected from L-tryptophan aminotransferases (EC 2.6.1.27) and tyrosine aminotransferases (aromatic) (EC 2.6.1.5) and L-amino acid oxidases (EC 1.4.3.2), and mutants derived from enzymes that have aspartate aminotransferase activity.

Example 16 identifies a specific enzyme, a mutant HEXaspC polypeptide that includes a substitution of Pro 9 for Tyr and a substitution of Arg 122 for Gly useful to facilitate the reactions of L-tryptophan and a-KG, oxaloacetate, pyruvate or combinations of them. themselves to form indole-3-pyruvate and L-glutamate, L-aspartate and L-alanine, respectively. Another specific enzyme that has "limited" activity is TatA, the S. meliloti tryptophan aminotransferase. Other suitable enzymes for the tryptophan reaction according to preferred embodiments of the pathway shown in Figure 1 include those with the following characteristics: an enzyme that transaminates MP at 1/10 the rate or less than the rate of L-tryptophan as in Example 16 or an enzyme when used with a racemase, as in Example 18, which produces more than 90% of the 4R isomers of monatin.

Examples of enzymes that do not have higher specificity for the conversion of L-tryptophan to indole-3-pyruvate compared to the conversion of MP to monatin include: HEXAspC (Example 16), Leishmania major broad specificity aminotransferase (WO 03/091396 A2), porcine aminotransferase (WO 03/091396 A2) and TatA from Rhodobacter sphaeroides (Example 18). However, these enzymes can evolve through mutagenesis for example to have limited activity for R-MP and / or (R, R) -monatin with respect to tryptophan.

If we now focus on the second reaction identified in Figure 1, the choice of enzyme to facilitate the reaction of indole-3-pyruvate to form MP determines the relative amount of (R, R) -monatin with respect to (S , R) -monatin produced. In general, the greater the relative amount of R-MP relative to the S-MP produced, the greater the relative amount of (R, R) -monatin relative to the (S, R) -monatin produced (when a D -enzyme facilitates the reaction of MP to form monatin). When a monatin composition having the R, R form of monatin as its sole monatin component is desired, an enzyme that selectively produces R-MP as opposed to S-MP (an "R-specific enzyme") should be used. The aldolase activity polypeptides described herein are useful for selectively producing R-MP, as opposed to S-MP. Several examples of R-specific aldolase enzymes are demonstrated in Table 1, above, in Examples 4, 5 and 6, below, and in the Sequence Listing.

If we now focus on the last stage of the pathway identified in Figure 1, it is shown that the reaction of R-MP to form (R, R) -monatin is facilitated by a broad specificity D-aminotransferase, for example D- alanine aminotransferase (EC 2.6.1.21, which is also known as D-amino acid aminotransferase or D-aspartate aminotransferase) or a D-amino acid dehydrogenase. As described above, the conversion of MP to monatin is an amination reaction, which creates a chiral center at the C-4 carbon of monatin. Where the R-chiral form is desired at the C-4 position, enzymes that produce chiral "R" centers in amino acids should be used.

According to some cases, D-aminotransferase has higher specificity, higher activity, or both for R-MP than for indole-3-pyruvate. According to some embodiments, D-aminotransferase has limited activity for indole-3-pyruvate. Enzymes with such characteristics can evolve or mutate from existing enzymes, for example, as shown in Example 16.

Examples 9 to 12 illustrate the production of (R, R) -monatin from D-tryptophan.

Figure 2 illustrates a production method of (R, R) -monatin and (S, R) -monatin. While in the Figure 1 embodiment, the aldolase used in the indole-3-pyruvate reaction to form R-MP affects the ratio of R, R: S, R formed, in the Figure 2 embodiment, the enzyme D that facilitates the conversion of MP to monatin determined the ratio between R, R: S, R formed. According to the pathway in Figure 2, if a non-stereospecific enzyme is used to facilitate the conversion of indole-3-pyruvate to MP, then both S-MP and R-MP can be formed. If a non-stereoselective aldolase is used to convert indole-3-pyruvate to MP, a stereoselective transaminase is required to convert the MP to (R, R) -monatin or (S, R) -monatin. As shown in Figure 2, the use of a Daminotransferase or D-amino acid dehydrogenase that is stereospecific for R-MP results in the production of (R, R) -monatin.

Figure 3 illustrates another alternative way to target (R, R) -monatin production. The pathway in Figure 3 is a modification of the pathway in Figure 1, where indole-3-pyruvate is produced indirectly, rather than directly, from L-tryptophan. More specifically, L-tryptophan is converted to D-tryptophan, and D-tryptophan is then converted to indole-3-pyruvate.

The conversion of L-tryptophan to D-tryptophan can be facilitated by means of a tryptophan racemase or functional equivalent thereof. Example 15 provides potential sources of tryptophan racemases and detection methods to identify such enzymes. It is also contemplated that a tryptophan racemase can evolve (such as by mutagenesis or recombinant manipulation) for better performance from an existing racemase amino acid.

Non-exhaustive examples of tryptophan racemases include homologs or amino acid mutants racemases (EC 5.1.1.-), for example serine racemase, where the homologs or mutants are capable of converting L-tryptophan to D-tryptophan. Non-exhaustive examples of sources from which the amino acid racemase may be derived include: microorganisms such as Salmonella typhimurium, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Schizosaccaroyces pombe, Bacillus cereus, Enterococcus gallinarum, Pediococcus, Pediococcus, Pediococcus , Lactobacillus brevis, Aquifex pyrophilus, Lactobacilli, Streptococcus, Anabaena sp., Pseudomonas striata, Lentinus edodes, Scapharca brouhtonii Desulfurococcus sp., Thermococcus sp. and Pseudomonas striata. Additional non-exhaustive examples of sources from which the amino acid racemase may be derived include silkworm, rat brain, or mouse brain.

Non-limiting examples of potential sources from which suitable tryptophan racemases can be derived include: microorganisms such as Pseudomonas, for example, Pseudomonas chlororaphis (Pseudomonas aurereofaciens) (ATCC 15926) and Burkholderia pyrrocina (ATCC15958). Additional non-limiting examples of potential sources from which suitable tryptophan racemases can be derived include plants, for example tobacco plants, such as Nicotiana tabacum, wheat plants, such as, Triticum aestivum, beets, tomatoes and Sclerochiton ilicifolius.

The route shown in Figure 3 has certain benefits, including that even when (R, R) -monatin is the desired product, the same enzyme can be used for the reaction that produces indole-3-pyruvate as for the reaction that produces monatin. That is, in the pathway illustrated in Figure 1, an L-aminotransferase (or suitable L-enzyme) facilitates the reaction that produces indole-3-pyruvate, but a D-aminotransferase facilitates the reaction that produces monatin. In contrast, in the pathway of Figure 3, a certain D-aminotransferase that facilitates the reaction that produces indole-3-pyruvate, can also facilitate the reaction that produces monatin. Accordingly, in the routes according to Figure 3, broad specificity D-aminotransferases may be preferred when it is desired to use the same enzyme for the indole-3-pyruvate-forming reaction as for the monatin-forming reaction. In contrast, in the pathways according to Figures 1, 2, 4, 6, 7 and 8, monatin production can proceed more efficiently when selecting a D-aminotransferase that has limited activity and / or specificity for indole -3 pyruvate compared to R-MP.

Another benefit of the pathway schematically depicted in Figure 3 is that the amino acid product of the reaction coupled to the indole-3-pyruvate-producing reaction can now be used as a starting material in the reaction coupled to the monatin-producing reaction. That is, in the route illustrated in Figure 1, L-tryptophan reacts to produce indole-3-pyruvate and at the same time oxaloacetate, alpha-ketoglutarate and / or pyruvate react to produce an L-amino acid. Since the reaction of R-MP to form monatin couples with a reaction that uses a D-amino acid as a substrate, the L-amino acid of the indole-3-pyruvate-forming reaction is not recycled, under the conditions shown , for use in the reaction coupled to the R-MP reaction. In contrast, in the pathway illustrated in Figure 3, the reaction of D-tryptophan to form indole-3-pyruvate couples with a reaction that forms a D-amino acid product. Said D-amino acid can be recycled for use in the reaction coupled to the R-MP reaction. This allows the use of non-stoichiometric amounts of an amino acceptor in step one. In some cases of the invention, the D-amino acid is D-alanine.

Figures 4 and 5 illustrate further modifications of the pathway shown in Figure 1, the modifications of which are aimed at recycling the amino acid product formed by the reaction coupled to the reaction of L-tryptophan with the amino acid reagent of the reaction coupled to the MP reaction with monatin.

With reference to Figure 4, recycling is accomplished by providing an enzyme that can facilitate the conversion of an L-amino acid to a D-amino acid and vice versa. More specifically, where as shown in Figure 4, a-KG reacts to form L-glutamate when L-tryptophan reacts to form indole-3-pyruvate, a glutamate racemase (EC 5.1.1.3) or functional equivalent can be provided which It can facilitate the conversion of Lglutamate to D-glutamate and vice versa. In that case, the L-glutamate formed together with the production of indole-3-pyruvate is removed in by its conversion to D-glutamate, and the D-glutamate formed from the conversion of Lglutamate is then available as a substrate for the reaction. coupled with the MP to the monatin reaction. Similarly, the a-KG formed in the D-glutamate reaction is available as a substrate for the reaction coupled to the reaction of L-tryptophan with indole-3-pyruvate.

Non-exhaustive examples of potential sources from which glutamate racemase may be derived include Pediococcus pentosaceus, Bacillus pumilus, Lactobacillus fermenti, Lactobacillus brevis, E. coli, Aquifex pyrophilus, and Bacillus subtilis. More specifically (also by way of non-limitation), glutamate racemase can be expressed from a nucleic acid such as the pediococcus pentaosaceus murI gene (Genbank Accession No. L22789), or glutamate racemase from Lactobacillus brevis.

In the case where oxaloacetate reacts to form L-aspartate when L-tryptophan reacts to form indole-3-pyruvate, an aspartate racemase (EC 5.1.1.13) or functional equivalent can be provided to convert L-aspartate to D- aspartate. In that case, the L-aspartate together with the production of indole-3-pyruvate is removed in by its conversion to D-aspartate, and the D-aspartate formed from the conversion of L-aspartate is then available as a substrate for the reaction coupled to the reaction of MP with monatin. Similarly, the oxaloacetate formed in the D-aspartate reaction is available to act as a substrate for the reaction coupled to the reaction of L-tryptophan with indole-3-pyruvate.

Non-exhaustive examples of suitable enzymes having aspartate racemase activity include ASPR-101 (BioCatalytics, Inc., Pasadena, CA) and homologs or mutants of an amino acid racemase (EC 5.1.1.-) that is capable of facilitating the conversion of L-aspartate to D-aspartate.

Non-exhaustive examples of potential sources from which aspartate racemases may derive include: Desulfurococcus, Thermococcus, bivalve mollusc Scapharca brouhtonii, Acinetobacter, Agrobacterium, Archaeoglobus, Bacillus, Closdetella, Bradyrhizobium, Brekibacterium, Burkbacterium, Burkbacterium, Burkibacterium, Burkbacterium, Burkibacterium, Burkbacterium, Burkibacterium, Burkbacterium, Caviar, Cactus Desulfitobacterium, Desulfotalea, Enterococcus, Erwinia, Escherichia, Ferroplasma, Helicobacter, Klebsiella, Lactobacillus, Mannheimia, Medicago, Mesorhizobium, Methanococcus, Oceanobacillus, Oenococcus, Pediococcus, Polaris Streptococcus, Thermoanaerobacter, Vibrio, Wolinella, Xanthomonas, Xanthobacter, Yersinia and Zymomonas.

In cases where pyruvate reacts to form L-alanine when L-tryptophan reacts to form indole-3-pyruvate, an alanine racemase or functional equivalent can be provided to convert L-alanine to Dalanin. In that case, the L-alanine formed together with the production of indole-3-pyruvate is removed by its conversion to D-alanine, and the D-alanine formed from the conversion of L-alanine is then available to act as a substrate for the reaction coupled to the reaction of MP with monatin. Similarly, the pyruvate formed in the D-alanine reaction is available to act as a substrate for the L-tryptophan-coupled reaction to the indole-3-pyruvate reaction.

Non-exhaustive examples of suitable alanine racemases include A8936 (Sigma-Aldrich, St. Louis, MO).

Non-exhaustive examples of potential sources from which alanine racemase may be derived include: Brucella

abortus,

Streptococcus faecalis Salmonella typhimurium,  Escherichia coli, Bacillus  subtilis, Bacillus

stearothermophilus,

Pseudomonas aeruginosa,  Vibrio  cholerae, Schizosacc aroyces pombe, Bacillus

cereus and Lentinus edodes.

Examples 18 and 21 illustrate the use of the above racemases, their impact on increasing the ratio of the desired monatin product, and provide potential sources for the racemase enzymes.

Referring to Figure 5, a stereoinvestment aminotransferase is used to facilitate the reaction of R-MP to form monatin. Although typically the reaction of R-MP (or S-MP) to form (R, R) -monatin (or (S, R) monatin) is coupled with the reaction of a D-amino acid, a steroinversion aminotransferase it can facilitate the coupled reactions of R-MP (or S-MP) to form (R, R) -monatin (or (S, R) -monatin) using a Lamino Acid. In this way, the L-amino acid product of the L-tryptophan aminotransferase reaction can be used as a substrate for transamination of MP to monatin, and the product (i.e., oxaloacetate, pyruvate, and / or a-KG) of the reaction coupled to the reaction of MP with monatin can be used as a starting material for the reaction coupled to the reaction of L-tryptophan with indole-3-pyruvate. Non-exhaustive examples of stereoinversion aminotransferases that can be used include D-phenylglycine aminotransferase (EC 2.6.1.72, also known as D-4-hydroxyphenylglycine aminotransferase) and D-methionine aminotransferase (EC 2.6.1.41, also known as D-met-aminotransferase) and D-methionine-pyruvate aminotransferase). Non-exhaustive examples of potential sources from which D-phenylglycine aminotransferase can be derived include Pseudomonas, such as Pseudomonas putida LW-4 and Pseudomonas stutzeri ST-201. Non-exhaustive examples of potential sources from which D-methionine aminotransferase can be derived include cauliflower and peanuts.

Examples 19 and 20 together provide potential sources of stereoinvestment enzymes, and methods of making such enzymes. The examples also provide detection methods to identify such enzymes. It is also contemplated that such enzymes can evolve from stereoinvestment enzymes that are known or found in nature. As a non-exhaustive example, the stereoinvestment aminotransferase can be a homolog or mutant of a D-amino acid aminotransferase or a homolog or mutant of an amino acid racemase (EC 5.1.1.-).

Figures 6-8 also illustrate modifications to the pathway of Figure 1. The pathways illustrated in Figures 6-8 provide methods for driving equilibrium reactions by removing the by-product of the tryptophan reaction and in some cases providing a substrate for the reaction. of MP.

Referring to Figure 6, the route shown removes the L-amino acid product from the reaction coupled to the tryptophan reaction by converting it to a different L-amino acid, and then provides a substrate for the reaction coupled to the MP reaction by converting the newly formed L-amino acid into a D-amino acid. Specifically, L-tryptophan is shown to react together with oxaloacetate to form indole-3-pyruvate and L-aspartate. An aspartate 4-decarboxylase (EC 4.1.1.12) or functional equivalent is used to facilitate the conversion of L-aspartate to L-alanine and carbon dioxide, and an enzyme with alanine racemase activity is used to facilitate the conversion of L- Alanine to D-Alanine. Said D-alanine can serve as an amino donor for the conversion of R-MP to monatin.

With respect to Figure 7, the route shown illustrates additional methods for removing the Lamino Acid product from the reaction coupled to the tryptophan reaction. Embodiments as presented in the figure produce a by-product that is not available to react in the reverse direction, for example due to volatility (such as carbon dioxide) or by spontaneous conversion into an unreacted final product. An example of such an approach includes cases where a-KG reacts together with L-tryptophan to produce Lglutamate, a glutamate decarboxylase (EC 4.1.1.15) or functional equivalent can be provided that can facilitate the conversion of L-glutamate to 4 -aminobutanoate (with carbon dioxide as a by-product). Non-exhaustive examples of potential sources from which L-glutamate decarboxylase can be derived include: Clostridium perfringens, C. welchii or E. coli.

Another example of such an approach to continue the tryptophan reaction includes where oxaloacetate reacts together with L-tryptophan, an aspartate decarboxylase (EC 4.1.1.1.1) or functional equivalent can be provided to facilitate the conversion of L-aspartate to -alanine ( with carbon dioxide as a by-product).

Referring to Figure 8, the route shown illustrates additional methods of removing the Lamino Acid product from the reaction coupled to the tryptophan reaction and providing a substrate for the reaction coupled to the MP reaction. Specifically, in cases where a-KG reacts together with L-tryptophan to form L-glutamate, an enzyme with L-alanine aminotransferase and pyruvate activity may be provided, where the enzyme L-alanine aminotransferase facilitates the pyruvate reaction. and L-glutamate to form L-alanine. An alanine racemase or functional equivalent may also be provided to facilitate the conversion of Lalanine to D-alanine. Said D-alanine can be used as a substrate together with MP to form monatin and pyruvate. See Examples 18 and 21.

Biosynthetic pathways to produce R, R and other stereoisomers of monatin derivatives

The methods of the disclosed invention using aldolase-active polypeptides described herein can be used to facilitate the reaction between a substituted indole-3-pyruvate and a C3 carbon source as defined in the claims.

Useful enzymes to facilitate a reaction between a substituted indole-3-pyruvate and a C3 carbon source include one or more aldolase-active polypeptides of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO : 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 , SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO : 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106 , SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 11 6, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ IDNO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof having aldolase activity.

In one embodiment, one or more polypeptides with HMG aldolase activity of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, I KNOW THAT ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO : 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162 , SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO : 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212 , SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO : 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262 , SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO : 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304 or fragments or subsequences thereof that have aldolase activity may be useful in facilitating a reaction between an indole-3 -substituted pyruvate and a C3 carbon source as further defined in the claims.

In another embodiment, one or more polypeptides with KHG aldolase activity of any of SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof having aldolase activity may be useful in facilitating a reaction between a substituted indole-3-pyruvate and a C3 carbon source as further defined in the claims.

Alternatively, one or more polypeptides with aldolase activity encoded by a nucleic acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or more, or full sequence identity (100%) with a nucleic acid, including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105 , SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO : 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155 , SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO : 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205 , SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID N O: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 can be useful to facilitate a reaction between an indole-3-pyruvate and a C3 carbon source as defined in the claims.

One or more polypeptides with HMG aldolase activity encoded by a nucleic acid sequence having at least about 95%, 96%, 97%, 98%, 99% or more, or full sequence identity (100%) with a nucleic acid, including SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 , SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO : 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157 , SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO : 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207 , SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305 can be helpful in facilitating a reaction between an indole-3-pyruvate and a C3 carbon source as further defined in the claims.

One or more polypeptides with KHG aldolase activity encoded by a nucleic acid sequence having at least about 95%, 96%, 97%, 98%, 99% or more, or full sequence identity (100%) with a nucleic acid including SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321 , SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 may be useful in facilitating a reaction between an indole-3-pyruvate and a C3 carbon source as further defined in the claims.

One or more polypeptides with aldolase activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, I KNOW Q ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 may be useful in facilitating a reaction between a substituted indole-3-pyruvate and a C3 carbon source as defined in the claims.

One or more polypeptides with HMG aldolase activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO : 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 , SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO : 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 , SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO : 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151 , SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO : 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201 , SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO : 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251 , SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 283, SEQ ID NO : 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 SEQ ID NO: 303, SEQ ID NO: 305 may be useful in facilitating a reaction between a substituted indole-3-pyruvate and a C3 carbon source as further defined in the claims.

In another embodiment of the invention, one or more polypeptides with KHG aldolase activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid of SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 and SEQ ID NO: 338 can be helpful in facilitating a reaction between a indole-3-pyruvate and a C3 carbon source as further defined in the claims.

In one embodiment, the substituent group for the substituted indole-3-pyruvate is a halogen atom attached to any carbon atom in the indole ring. In another embodiment, the substituent group is a chlorine atom attached to any carbon in the indole ring. In yet another embodiment, the monatin derivative is 4-hydroxy-4- (6-methylindole-3-methylmethyl) glutamic acid.

Polypeptides that have aldolase activity can be used in a route in which one or more steps is a chemical synthesis reaction. For example, in some embodiments, one or more polypeptides that have aldolase activity can facilitate a reaction between pyruvate and indole-3-pyruvate to provide monatin precursor. The monatin precursor can then be purified. A reductive amination reaction of the monatin precursor can then be used to provide monatin.

Polypeptides having aldolase activity as well as other enzymes used in the process to produce monatin and monatin derivatives can be used in pure, crude, isolated or suspension form of ammonium sulfate.

Polypeptides that have aldolase activity can be optimized using stabilizing agents, including dithiothreitol ("DTT") and -mercaptoethanol.

The monatin or monatin derivative that is produced using one or more of the polypeptides disclosed herein, is generally at least about 50 to about 99% (R, R) -monatin or (R, R) -monatin derivative, by weight of the monatin or total monatin derivative produced. In other embodiments, the monatina

or monatin derivative produced using one or more of the polypeptides disclosed herein, is greater than 60% (R, R) -monatin or (R, R) -monatin derivative, by weight of the total monatin produced; for example, the (R, R) monatin or derivative of (R, R) -monatin is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%

or 99% of the monatin or total monatin derivative produced. Alternatively, various amounts of two or more preparations of monatin or monatin derivatives can be combined so as to result in a preparation that is a desired percentage of (R, R) -monatin or derivative of (R, R) -monatin. For example, a monatin preparation that is 60% (R, R) -monatin can be combined with a monatin preparation that is 90% (R, R) -monatin; if equal amounts of 60% and 90% of (R, R) -monatin are combined, the resulting monatin preparation would be 75% (R, R) -monatin.

Monatin or monatin derivative, or an intermediate (including a monatin precursor), produced using one or more polypeptides disclosed herein, can be purified from the components of the reaction. In one embodiment, monatin, monatin derivative or intermediate, such as monatin precursor, can be purified simply by removing the substance to be purified from the enzyme preparation in which it is synthesized.

In other embodiments, the monatin intermediate, precursor, monatin, or monatin derivative is purified from the preparation in which it was synthesized such that the resulting "purified" composition or preparation is at least about 5-60% monatin in weight of total organic compounds. In another embodiment, monatin, monatin derivative or intermediate, such as a monatin precursor, can be purified to a degree of purity of at least about 70%, 80%, 90%, 95% or 99% by weight of the compounds total organic. Monatin, derived from monatin or the intermediate (including monatin precursor), produced using one or more of the polypeptides disclosed herein, can be purified from the reaction components by any method known to one skilled in the art. technique. Optimally, the purified monatin or intermediate can be repeatedly recrystallized until the desired degree of purity is achieved.

The following examples are offered to illustrate, without limitation, the claimed invention.

EXAMPLES

Example 1

Detection of monatin, monatin precursor, tryptophan, alanine, aspartate and glutamate The present example describes the procedures used to detect the presence of monatin, monatin precursor (PM), tryptophan, aspartate, alanine and glutamate. Also, the present example describes a procedure for separating and detecting the four monatin stereoisomers.

Monatin and Tryptophan LC / MS / MS Multiple Reaction Monitoring (MRM) Analysis

Analyzes of mixtures for monatin and tryptophan derived from in vitro or in vivo biochemical reactions were performed using a Waters Micromass liquid chromatography (LC / MS / MS) tandem mass spectrometry instrument including a 2795 liquid chromatograph. from Waters with a Waters 996 Photodiode Absorbance Monitor (PDA) placed in series between the chromatograph and a Micromass Quattro Ultima Triple Quadrupole Mass Spectrometer. LC separations were performed using a 2.1mm x 250mm Xterra MS C8 reverse phase chromatography column at 40 ° C. The LC mobile phase consisted of A) water containing (i) 0.05% (v / v) trifluoroacetic acid or (ii) 0.3% formic acid and 10 mM ammonium formate and B) methanol with (i) 0.05% (v / v) trifluoroacetic acid or (ii) 0.3% formic acid and 10mM ammonium formate.

If the LC mobile phase consisted of A) water with 0.05% (v / v) trifluoroacetic acid and B) methanol with 0.05% (v / v) trifluoroacetic acid, the gradient elution was linear from 5% B to 35% B, for 0-4 minutes, linear from 35% B to 60% B, for 4-6.5 minutes, linear from 60% B to 90% B, for 6 , 5-7 minutes, isocratic at 90% B, for 7-11 minutes, linear from 90% B to 95% B, for 11-12 minutes, linear from 95% B to 5% B, for 12 -13 minutes, with a rebalancing period of 2 minutes between sets. The flow rate was 0.25 mL / min and a PDA absorbance of 200nm to 400nm was monitored. All ESI-MS parameters were optimized and selected based on the generation of protonated molecular ions ([M + H] +) from the analytes of interest, and the production of ions from characteristic fragments. The following instrumental parameters were used for the monatin and tryptophan LC / MS / MS Multiple Reaction Monitoring (MRM) analysis: Capillary: 3.5 kV; Cone: 40V; Hex 1:20 V; Aperture: 0V; Hex 2: 0 V, Source Temperature: 100ºC; desolvation temperature; 350 ° C; Desolvation gas: 500 L / h; Gas cone: 50 L / h; Low mass resolution (Q1): 12.0; High mass resolution (Q1): 12.0; Ionic energy: 0.2; Input: -5V; Collision energy: 8; Output: IV; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ionic energy (Q2): 3.5; Multiplier: 650. Five specific monatin father / daughter MRM transitions are used to specifically detect monatin reactions in vitro and in vivo. The monitored transitions are 293.1 to 158.3, 293.1 to 168.2, 293.1 to 211.2, 293.1 to 230.2 and 293.1 to 257.2. Tryptophan is monitored with the MRM transition 204.7 to 146.4. For internal standard quantification of monatin and tryptophan, four calibration standards were analyzed with four different proportions of each analyte with respect to d5-tryptophan and d5-monatin. These data are subjected to a linear least squares analysis to form a calibration curve for monatin and tryptophan. A fixed amount of d5-tryptophan and d5-monatin is added to each sample (d5-monatin was synthesized from d5-tryptophan according to the procedures of patent WO03 / 091396A2) and the response ratios (monatin / d5-monatin ; tryptophan / d5-tryptophan) used in conjunction with the calibration curves described above to calculate the amount of each analyte in the mixtures.

If the LC phase was A) water with 0.3% formic acid and 10 mM ammonium formate and B) methanol with 0.3% formic acid and 10 mM ammonium formate, the gradient elution it was linear from 5% B to 45% B, 08.5 minutes, linear from 45% B to 90% B, 8.5-0 minutes, isocratic from 90% B to 90% B, 9 -12.5 minutes, linear from 95% B to 5% B, 12.5-13 minutes, with a rebalancing period of 4 minutes between sets. The flow rate was 0.27mL / min, and the PDA absorbance was monitored from 210nm to 400nm. All ESI-MS parameters were optimized and selected based on the generation of protonated molecular ions ([M + H] +) from the analytes of interest and the production of ions from characteristic fragments. The instrumental parameters used for this secondary mobile phase are the same as the previous parameters. Four monatin-specific father-daughter MRM transitions and one specific father / daughter tryptophan transition are used to specifically detect monatin and tryptophan reactions in vivo and in vitro. The monitored transitions are 293.1 to 158.0, 293.1 to 168.0, 293.1 to 211.5 and 293.1 to 257.0. Tryptophan is monitored with the MRM 205.2 to 146.1 transition. For internal standard quantification of monatin and tryptophan, four calibration standards containing four different ratios of each analyte to d5-tryptophan and d5monatin were analyzed. These data are subjected to a linear least squares analysis to form a calibration curve for monatin and tryptophan. A fixed amount of d5-tryptophan and d5-monatin is added to each sample (d5monatin was synthesized from d5-tryptophan according to the procedures of patent WO03 / 091396 A2), and the response ratios (monatin / d5 are used -monatin; tryptophan / d5-tryptophan) together with the calibration curves described above to calculate the amount of each analyte in the mixtures. The monitored daughter-father mass transitions for d5-tryptophan and d5-monatin are 210.2 to 151.1, and 298.1 to 172.0, respectively.

Exact measurement of monatin mass

High resolution MS analysis was performed using a Q-Star hybrid quadrupole / time-of-flight mass spectrometer from Applied Biosystems-Perkin Elmer. The mass measured for the protonated monatin used tryptophan as the internal mass calibration standard. The calculated mass of protonated monatin, based on the basic composition C14H17N2O5, is 293.1137. Monatin produced using the biocatalytic process described in Examples 2 and 3 showed a measured mass of 293.1144. This is a mass measurement error of less than 2 parts per million (ppm), which provides conclusive proof of the basic composition of monatin produced enzymatically.

Monatin chiral LC / MS / MS (MRM) measurement.

The determination of the distribution of monatin stereoisomers in in vitro and in vivo reactions was achieved by derivatization with 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (FDAA), followed by MRM LC / MS / measurement. Reverse phase MS.

Monatin derivatization with FDAA

In a 50 µL sample or standard and an internal 10 µL standard, 100 µL or 200 µL of 1% FDAA solution in acetone were added. Twenty or forty µL of 1.0 M sodium bicarbonate were added, respectively, and the mixture was incubated for 1 hour at 40 ° C with occasional mixing. The sample was removed and cooled and neutralized with 20 µL of 2.0M HCI (more HCI may be required to neutralize a buffered biological mixture). After degassing was complete, the samples were ready for analysis by LC / MS / MS.

Monitoring of LC / MS / MS multiple reaction for determination of monatin stereoisomer distribution in in vitro and in vivo reactions

Analyzes were performed using the previously described LC / MS / MS instrumentation. LC separations capable of separating the four monatin stereoisomers (specifically FDAA-monatin) were performed on a 2.0 x 250 mm (3 µm) reverse phase chromatography column of Phenomenex Luna C18 (2) at 40 ° C. The LC mobile phase consisted of A) water with 0.05% (mass / volume) of ammonium acetate and B) acetonitrile. Elusion was isocratic at 13% B, 0-2 minutes, linear from 13% B to 30% B, 2-15 minutes, linear from 30% B to 80% B, 15-16 minutes, isocratic at 80% B, 16-21 minutes, and linear from 80% B to 13% B, 21-22 minutes with a rebalancing period of 8 minutes between sets. The flow rate was 0.23 mL / min, and the PDA absorbance was monitored from 200nm to 400nm. All ESI-MS parameters were optimized and selected on the basis of a generation of unprotonated molecular ions ([M − H] -) of FDAA-monatin, and the production of characteristic fragment ions.

The following instrumental parameters were used for monatin LC / MS analysis in negative ion ESI / MS mode: Capillary: 2.0 kV; Cone; 25 V; Hex 1:10 V; Opening; 0 V; Hex 2: 0 V; Source temperature: 100ºC; desolvatation temperature: 350 ° C; Desolvation gas: 500 L / h; Gas cone: 50 L / h; Low mass resolution (Q1): 12.0; High mass resolution (Q1): 12.0; Ionic energy: 0.2; Input: -5V; Collision energy: 20; Output: 1V; Low mass resolution (Q2): 12; High mass resolution (Q2): 12; Ionic energy (Q2): 3.0; Multiplier: 650. Three specific father-daughter monatin-FDAA transitions are used to specifically detect FDAA-monatin reactions in vitro and in vivo. The monitored transitions for monatin are 543.2 to 268.1, 543.2 to 499.3, and 543.2 to 525.3. The mass transition derived from the monatin internal standard was 548.2 to 530.3. Identification of FDAA-monatin stereoisomers is based on chromatographic retention time compared to purified synthetic monatin stereoisomers and mass spectral data. An internal standard is used to monitor the progress of the reaction and to confirm the retention time of the S, S stereoisomer.

Post-column fluorescence detection with liquid chromatography of amino acids including glutamate and alanine.

Liquid chromatography with post column fluorescence detection (LC / OPA) for the determination of glutamate and alanine in in vivo and in vitro reactions was performed on a Waters 2690 LC system or equivalent combined with a fluorescence detector by scanning 474 and a Waters post column reaction module. Semi-quantitative analyzes of monatin and tryptophan were also performed using this procedure. LC separations were performed on a sodium interaction charged ion exchange column at 60 ° C. Mobile phase A consisted of Pickering Na 328 buffer (Pickering Laboratories, Inc.; Mountain View, CA) Mobile phase B consisted of Pickering Na 740 buffer. Gradient elution was from 0% to 100% B, 0-20 minutes, isocratic at 100% B, 20-36 minutes and linear from 100% B to 0% B, 36-37 minutes, with at least a 5 minute rebalancing period between segments, depending on the sample matrix. The flow rate for the mobile phase was 0.5 mL / min. The flow rate for the OPA post-column derivatization solution was 0.5 mL / min. The fluorescence detector settings were EX 338-340 nm and Em 420-425 nm. Norleucine was used as an internal standard for the analysis. Amino acid identification was based on chromatographic retention time data for purified standards.

Detection of L- and D-amino acids by LC / MS / MS

Samples containing a mixture of L- and D-amino acids such as lysine, alanine, methionine, tyrosine, leucine, phenylalanine, tryptophan, glutamate, and aspartate obtained from biochemical reaction experiments were first treated with formic acid to denature the protein . The sample was then centrifuged and filtered through a 0.45 µm nylon syringe filter prior to LC / MS / MS analysis. The identification of L- and Daminoacids was based on retention time and selective mass detection. LC separation was accomplished using a Waters 2690 liquid chromatography system and an ASTEC 2.1mm x 250mm Chirobiotic TAG chromatography column with a column temperature of 45 ° C. Mobile phases A and B of the LC consisted of 0.25% acetic acid and 0.25% acetic acid in methanol, respectively. Isocratic elution was used for all procedures to separate the L and D isomers. Lysine was eluted using 80% mobile phase A and 20% mobile phase B. Glutamate, alanine and methionine were separated with the elution of 60% of mobile phase A and 40% of mobile phase B and a flow rate of 0.25 mL / min. Aspartate, tryptophan, tyrosine, leucine, and phenylalanine were separated isomerically with 30% of mobile phase A and 70% of mobile phase B with a flow rate of 0.3 mL / min for all minus phenylalanine, which was used at a flow rate of 0.25 mL / min.

The detection system for analysis of L- and D-amino acids included a Waters 996 photodiode detector (PDA) and a Micromass Quattro Ultima triple quadrupole mass spectrometer. The PDA, which scans from 195 to 350 nm, was placed in series between the chromatography system and the mass spectrometer. The parameters for the Micromass Quattro Ultima triple quadrupole mass spectrometer operating in positive electrospray ionization (+ ESI) mode were as follows: Capillary: 3.0 kV; Cone: 20V; Hex 1:15 V; Aperture: 1V; Hex 2: 0 V: Source temperature. 100 ° C; desolvatation temperature: 350ºC; desolvation gas: 530 L / h; gas cone: 30 L / h; Low mass resolution Q1: 12.5; High mass resolution Q1: 12.5; Ionic energy 1: 0.2; Input: -5; Collision: 8; Exit 1: 10; Low mass resolution Q2: 12.5; High mass resolution Q2: 12.5; Ionic energy 2: 0.5; Multiplier: 650 V. MS / MS experiments with a Multiple Reaction Monitoring (MRM) mode were set up to selectively monitor reaction transitions from 147.8 to 84.2 and from

Glutamate 147.8 to 102.1, 134.00 to 74.30 and 134.00 to 88.2 for aspartate, 147.3 to 85.0 for lysine, 150.3 to 104.8 for methionine, 182 , 3 to 137.0 for tyrosine, 132.3 to 87.0 for leucine and 166.3 to 121.0 for phenylalanine. When two transitions were listed, the last ones were used for their quantification. For tryptophan, MS / MS experiments were determined with the Multiple Reaction Monitoring (MRM) mode to selectively monitor reaction transitions from 205.2 to 118.2, 205.2 to 146.1, and 205 , 2 to 188.2 and the transition from 212.1 to 151.1 for d8-DL-tryptophan. Quantification of tryptophan was achieved by determining the ratio of the analyte response of the transition from 205.2 to 146.1 in the internal standard, d8-D, L-tryptophan. Alternatively, the quantification of tryptophan, glutamate, and aspartic acids was based on the signal responses of m / z = 146.5, m / z = 102.1, and m / z = 88.2, respectively,

Monatin and monatin precursor (MP) production for standards and assays

Monatin production

A racemic mixture of (R, R) -monatin and (S, S) -monatin was produced synthetically as described in US Patent No. 5,128,482.

(R, R) -monatin and (S, S) -monatin were separated by derivatization and hydrolysis. In synthesis, the racemic monatin mixture was esterified, the free amino group was blocked with Cbz, a lactone was formed, and the S, S lactone was selectively hydrolyzed using an immobilized protease enzyme. Monatin can also be separated as described in Bassoli, A. et al., Eur. J. Org. Chem., 8: 1652-1658 (2005).

MP production

R-MP was produced by transamination of (R, R) -monatin using a wide range D-aminotransferase AT-103 (BioCatalytics, Pasadena, CA) in 0.1M potassium phosphate buffer, using sodium pyruvate as the amino acceptor. S-MP was produced by transamination of (S, S) -monatin using an AT-102 Laminotransferase (BioCatalytics, Pasadena, CA) in a 0.1 M potassium phosphate buffer using sodium pyruvate as the acceptor of amino. Both reactions were performed at 30 ° C and at a pH of about 8.0-8.3 for about 20 hours. Both compounds were purified using a preparative HPLC scale with a hydrophobic resin (XADTM1600) from Rohm & Haas (Philadelphia, PA) eluting in water. Samples containing more than 90% pure monatin precursor were obtained and frozen to dry.

Example 2

Monatin precursor detection

The present example describes the procedures used to separate and detect the two enantiomers of the monatin precursor.

Non-chiral method for the detection of the monatin precursor

Reaction samples in 96-well plates were injected onto a 3.0 x 150mm, 3.5 µm column of Agilent Zorbax RX-C18 using a CTCPal Autosampler (LEAP Technologies, Carrboro, N.C.). The products were separated using an H20 / ACN gradient (0.1% formic acid):

LC-10ADvp pumps (Shimadzu, Kyoto, Japan) provided the gradient at 0.8 mL / min. Products were detected using an API4000 triple quadrupole mass spectrometer from Turbolon (Applied Biosystems, Foster City, CA). Ion vaporization and multiple ion monitoring was performed for analytes of interest in negative ion mode and each analysis lasted 6.5 minutes.

Pyruvate = 87.1 [M − H +] -

Indole-3-pyruvate = 202.1 [M − H +] -

Product = 290.0 [M − H +] -

Chiral analysis by EC of monatin precursors R and S

A P / ACE ™ MDQ capillary electrophoresis instrument (Beckman Coulter, Fullerton, CA) was used. The Chiral Development Kit was used, which includes small amounts of various chiral selectors, necessary buffers and 2 capillaries (Beckman, Fullerton, CA). Alternatively, for the MP assay only, the following reagents and other supplies may be obtained separately from Beckman Coulter (Fullerton, CA) or elsewhere:

N-CHO coated capillary; 50 µm ID, 65 cm full length or a fused silica capillary.

25 mM phosphate buffer, pH 5.

Hydroxypropyl 25 mg - cyclodextrin

A 10 ml hair conditioning solution (alternatively 0.5% polyethylene oxide solution, Mw, 600,000 or 300,000 daltons can be used)

Capillary Electrophoresis (CE) Analysis

A coated neutral capillary, 50 µM ID, 60 cm (50 cm for detection) or 30 (20) cm was used in conjunction with a DAD (or simple UV) detection at 214 nm. The separation capillary was heated to 15 ° C, and the samples to 4 ° C. The separation buffer consisted of 20 mM hydroxypropyl-cyclodextrin, 25 mM phosphate and a pH of 5. Sample injection was generally 0.5 psi, 5 s. The separation was performed at 500V / cm, reverse polarity (15 kV for a 30 cm capillary, 30 kV for 60 cm). The typical current used during the separation was -28! A. Typical migration times for the MP peaks were 3.5 minutes (20 cm effective length) or 8 minutes (50 cm).

An optional hair cleaning / washing / conditioning step prior to the sample runs used H2O for 4 minutes, 0.1M HCl for 1 minute, H2O for 1.5 minutes, hair conditioning solution for 4 minutes, H2O for 1 minute and separation buffer for 4 minutes.

A summary of the serial procedure was as follows: rinse the separation buffer for 1-2 minutes, sample injection 5 s at 0.5 psi, separation for 5-10 minutes at a reverse voltage polarity of 15 or 30 kV depending on capillary length.

Example 3

Pyruvate Aldolases General Assay

An exemplary procedure to measure the activity of different pyruvate aldolases uses a general substrate, 4-carboxy-4-hydroxy-2-oxoadipate (CHA). The CHA assay was adapted from the literature assays (eg, see E.E. Dekker & R.P. Kitson, J. Biol. Chem. 267, 10507-10514, 1992). A typical assay included 50 mM sodium phosphate with a pH of 7.5, 1 mM MgCl2, 1 mM CHA, 10 µg / ml Lactobacillus leichmanii D-lactate dehydrogenase (LDH) (Sigma-Aldrich, St. Louis , MO), 0.5 mM NADH. The assay started by incorporating enzyme (generally between 1 and 5 µL). Pyruvate release, along with NAD + formation, was permanently monitored on a spectrometer at 340 nm.

CHA was synthesized according to the procedure described in Tack, B.F. Chapman, P.J., and S. Dagley. J. Biol. Chem. 247 6438-6443 (1972).

A unit of enzyme activity, such as pyruvate aldolase, as HMG and / or KHG aldolase enzyme, is defined as the amount that pyruvate releases sufficient to reduce the absorbance at 340 nm by an OD of 1 to 5 minutes.

Example 4

Discovery of new keto-hydroxy-glutarate (KHG) and hydroxy-methyl-keto-glutarate (HMG) aldolases from the environmental libraries of Diversa

More than 150 unique HMG aldolases and 15 KHG aldolases were discovered by monitoring DNA libraries

10 of Diversa. These aldolase genes were sequenced and subcloned into a suitable expression vector. This vector was subsequently transformed into an expression host suitable for the production of sufficient amounts of aldolase for characterization of the enzyme. A selected set of aldolases were tested for activity in CHA and also for formation of a monatin precursor (MP). All of the enzymes discovered and described in this patent have potential for use in other bond-forming reactions.

Carbon-carbon between the alpha-keto acid acceptor and pyruvate or the pyruvate derived donor as exemplified in the general reaction scheme below.

Aldolasa

R = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl

R2 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl

R3 = H, alkyl, substituted alkyl, aryl, substituted aryl, benzyl, substituted benzyl, carboxylic acid

Example 5

Characterization of the selected aldolases

The selected aldolases were characterized with respect to their ability to catalyze the conversion of indole-3-pyruvate and pyruvate in the monatin precursor (MP) as shown in the following scheme:

Figures 13 and 14 show the activities of 58 different aldolases in the formation of the MP measured by LC / MS / MS.

The aldol reactions were performed with 20mM indole-3-pyruvate ("I3P"), 50mM pyruvate, 100mM sodium phosphate with a pH of 7.1mM MgCl2, 100 µg / mL of aldolase. Reactions were incubated at room temperature in the dark. The aliquots (30 µL) were removed several times and the reactions were stopped by storing the samples on ice. A portion of each aliquot was subjected to EC analysis while the remaining portion was diluted 1: 1000 in 50% acetonitrile and subjected to LC / MS analysis.

Table 2: Enantioselectivity of different aldolases for the formation of MP from I3P and pyruvate determined by chiral CE

Aldolasa

% of R-MP (23 hour deadline) CHA (U / mg) (based on total protein in lysate) Relative expression

SEQ ID NO: 28 (encoded by SEQ ID NO: 27)

98+ 31.8 ###

SEQ ID NO: 116 (encoded by SEQ ID NO: 115)

95 304 ##

SEQ ID NO: 76 (encoded by SEQ ID NO: 75)

fifty 424 ###

SEQ ID NO: 298 (encoded by SEQ ID NO: 297)

98+ 388 ##

SEQ ID NO: 44 (encoded by SEQ ID NO: 43)

70 332 ##

SEQ ID NO: 54 (encoded by SEQ ID NO: 53)

98+ 95 #

SEQ ID NO: 148 (encoded by SEQ ID NO: 147)

90 200 ##

SEQ ID NO: 46 (encoded by SEQ ID NO: 45)

70 174 ##

SEQ ID NO: 134 (encoded by SEQ ID NO: 133)

90 576 ###

SEQ ID NO: 142 (encoded by SEQ ID NO: 141)

98+ 55 #

SEQ ID NO: 122 (encoded by SEQ ID NO: 121)

98+ 38 ##

SEQ ID NO: 74 (encoded by SEQ ID NO: 73)

80 484 ###

SEQ ID NO: 64 (encoded by SEQ ID NO: 63)

95 38 #

SEQ ID NO: 108 (encoded by SEQ ID NO: 107)

98+ 40 #

SEQ ID NO: 796 (encoded by SEQ ID NO: 95)

98+ Not detected #

SEQ ID NO: 126 (encoded by SEQ ID NO: 125)

95 124 ##

SEQ ID NO: 80 (encoded by SEQ ID NO: 79)

98+ Not detected #

SEQ ID NO: 36 (encoded by SEQ ID NO: 35)

98+ 80 ##

Aldolasa

% of R-MP (23 hour deadline) CHA (U / mg) (based on total protein in lysate) Relative expression

SEQ ID NO: 62 (encoded by SEQ ID NO: 61)

98+ Not detected #

SEQ ID NO: 112 (encoded by SEQ ID NO: 111)

98+ Not detected #

SEQ ID NO: 130 (encoded by SEQ ID NO: 129)

98+ 38 ##

SEQ ID NO: 94 (encoded by SEQ ID NO: 93)

98+ 47 ##

SEQ ID NO: 102 (encoded by SEQ ID NO: 101)

Not detected Not detected #

SEQ ID NO: 58 (encoded by SEQ ID NO: 57)

98+ 59 ##

SEQ ID NO: 88 (encoded by SEQ ID NO: 87)

fifty 510 ###

SEQ ID NO: 50 (encoded by SEQ ID NO: 49)

98+ 144 ##

SEQ ID NO: 106 (encoded by SEQ ID NO: 105)

98+ Not detected ##

SEQ ID NO: 40 (encoded by SEQ ID NO: 39)

40 406 ###

SEQ ID NO: 42 (encoded by SEQ ID NO: 41)

98+ 92 ##

SEQ ID NO: 278 (encoded by SEQ ID NO: 277)

95 2.0 #

SEQ ID NO: 162 (encoded by SEQ ID NO: 161)

95 11.8 #

SEQ ID NO: 276 (encoded by SEQ ID NO: 275)

98+ 100.4 ####

SEQ ID NO: 178 (encoded by SEQ ID NO: 177)

95 38.8 #

SEQ ID NO: 202 (encoded by SEQ ID NO: 201)

Not detected Not detected #

SEQ ID NO: 166 (encoded by SEQ ID NO: 165)

98+ 85.5 ##

SEQ ID NO: 218 (encoded by SEQ ID NO: 217)

95 49.1 ##

SEQ ID NO: 224 (encoded by SEQ ID NO: 223)

98+ 23.2 #

SEQ ID NO: 226 (encoded by SEQ ID NO: 225)

98+ 128.3 #

Aldolasa

% of R-MP (23 hour deadline) CHA (U / mg) (based on total protein in lysate) Relative expression

SEQ ID NO: 244 (encoded by SEQ ID NO: 243)

98+ 40.4 #

SEQ ID NO: 250 (encoded by SEQ ID NO: 249)

95 6.0 #

SEQ ID NO: 252 (encoded by SEQ ID NO: 251)

95 20.2 ##

SEQ ID NO: 264 (encoded by SEQ ID NO: 263)

95 9.9 ##

SEQ ID NO: 268 (encoded by SEQ ID NO: 267)

95 2.0 #

SEQ ID NO: 272 (encoded by SEQ ID NO: 271)

95 6.7 #

SEQ ID NO: 184 (encoded by SEQ ID NO: 183)

95 Not detected #

SEQ ID NO: 282 (encoded by SEQ ID NO: 281)

05 36.7 ###

SEQ ID NO: 186 (encoded by SEQ ID NO: 185)

95 4.2 #

SEQ ID NO: 192 (encoded by SEQ ID NO: 191)

95 11.9 #

SEQ ID NO: 200 (encoded by SEQ ID NO: 199)

95 17.9 ##

SEQ ID NO: 280 (encoded by SEQ ID NO: 279)

fifty Not detected #

SEQ ID NO: 284 (encoded by SEQ ID NO: 283)

90 2.2 #

SEQ ID NO: 172 (encoded by SEQ ID NO: 171)

95 8.4 #

SEQ ID NO: 180 (encoded by SEQ ID NO: 179)

98+ 61.0 ###

SEQ ID NO: 168 (encoded by SEQ ID NO: 167)

98+ 9.3 #

SEQ ID NO: 228 (encoded by SEQ ID NO: 227)

98+ 38.7 ###

SEQ ID NO: 236 (encoded by SEQ ID NO: 235)

95 13.1 #

SEQ ID NO: 238 (encoded by SEQ ID NO: 237)

98+ 22.3 ##

SEQ ID NO: 240 (encoded by SEQ ID NO: 239)

95 Not detected #

Aldolasa

% of R-MP (23 hour deadline) CHA (U / mg) (based on total protein in lysate) Relative expression

SEQ ID NO: 270 (encoded by SEQ ID NO: 269)

40 4.6 #

SEQ ID NO: 156 (encoded by SEQ ID NO: 155)

98+ 133.0 ###

Please note that the selectivities for R-MP of 98 +% indicate that no S-MP was detected. Given the sensitivity of the CE assay, the results indicate that at least 98 +% of MP formed is the R enantiomer. Therefore, the enzymes indicated with 98 +% are at least 98% selective towards R-MP and can be up to

5 100% selective.

Table 2 also shows the activity of the enzymes in a general aldolase substrate, CHA, as well as the relative expression of each enzyme, as determined by SDS-PAGE. Please note that several enzymes did not show detectable activity in CHA, but did show activity by generating a MP.

In summary, aldolases display a wide range of activities, expressions, and selections. Also, there are 10 many aldolases that show highly fine selectivities (98% or more) for R-MP.

Example 6 (not claimed)

Discovery of pyruvate aldolases in plants

Degenerate PCR primers (see below) were designed and used to extract aldolase genes from cDNA prepared from Sclerochiton ilicifolius. The 5 'and 3' ends of the genes were retrieved and the genes in length

Total 15 were amplified by PCR.

Primer

AARGTBTWGARGACAATG (SEQ ID NO: 335)

GCDCAGAWCAAYGGRTGG (SEQ ID NO: 336)

CCATCRSYATCDGCRTADAGCCA (SEQ ID NO: 337)

GCRTADAGCCAYTCNCCRTC (SEQ ID NO: 338)

Example 7 (not claimed)

Cloning of Bacillus sphaericus D-amino acid aminotransferase

Bacillus sphaericus D-amino acid aminotransferase (EC 2.6.1.21 also known as D-alanine

Aminotransferase or D-aspartate aminotransferase) was produced recombinantly for use in combined assays with various aldolases. This enzyme is homologous to the D-aminotransferases previously described for the production of monatin (United States publication No. 20040063175 and United States publication No. 20050282260).

Strains

B. sphaericus (ATCC number 10208) was grown on nutrient agar at 30 ° C for 24 hours. Colony clusters were placed in 100 µL of sterile water and heated for 5 minutes at 95 ° C to alter the cells. Three µL were used in subsequent amplifications of Polymerase Chain Reactions (PCR).

Polymerase chain reaction protocol

Primers were designed to clone into vectors pET 28b and pET 30a (Novagen, Madison, WI), using the Ncol and BamHI sites. The pET 30 construct contains an N-terminal labeled with Histidine and Serine while the pET28 construct is unlabeled.

Bacillus sphaericus primers:

Terminal N: 5’-GATATACCATGGCATACTCATTATGGAATG-3 ’(SEQ ID NO: 383) and terminal C: 5’-GTTATCGGATCCTTAGGCATTAATTGAAATTG-3’ (SEQ ID NO: 384)

The coding region was amplified using the following PCR protocol. In a 50 µl reaction, 3 µl of template, 1.6 µl of each primer, 0.25 mM of each dNTP, 3.5 U of High Fidelity Polymerase (Roche, Indianapolis, IN) and a Expand ™ Buffer IX with Mg. The thermocycler program used included a warm start at 94 ° C for 3 minutes, followed by 8 repetitions of the following steps: 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 2 minutes. Twenty-two cycles were performed with a homogeneous temperature of 58 ° C. After 30 cycles, the sample was kept at 72 ° C for 7 minutes and was subsequently stored at 4 ° C. The correct size PCR-free products were obtained (approximately 850 bp for said DAT gene).

Cloning

The PCR products were purified using a Qiaquick PCR Purification Kit from Qiagen (Qiagen, Valencia, CA) and digested with BamHI and Ncol in BamHI buffer (New England Biolabs, Ipswich, MA).

The vector and the digested inserts were purified using the Qiaquick Gel Extraction Kit from Qiagen (Qiagen, Valencia, CA). Ligatures were performed using the Roche Rapid Ligation Kit (Roche, Indianapolis, IN) and purified using the QIAquick PCR Purification Kit. The ligatures were transformed into Escherichia coli from the DH10B strain using a 0.2 cm cuvette and a Bio Rad Gene Pulser II system as described in the Bio Rad electroporation manual (Bio Rad, Hercules, CA). Cells were recovered in 900 µL SOC for 30 minutes at 37 ° C at 225 rpm. Cells were plated on LB agar plates containing kanamycin (25 µg / mL).

Plasmid DNA was purified using a Qiagen spin miniprep kit (Qiagen, Valencia, CA) and monitored for the correct inserts for restriction digestion with BamHI and Ncol. Plasmid sequences that appeared to have the correct insert were verified through the dideoxy DNA strand termination sequence at Agencourt BioScience Corporation (Beverly, MA). The sequence verified the coding sequence found in NCBI Region order number AF081278: 134,985 (gi: 3513754), which produces a protein with an amino acid sequence as listed by order number AAC33964 (gi: 3513755).

Gene expression and assays

Plasmid DNA was subcloned into the E. coli BL21 (DE3) expression host (Novagen, Madison, WI). Cultures were grown and plasmids were isolated using the Qiagen miniprep kit (Qiagen, Valencia, CA) and analyzed by restriction digestion to confirm identity. Induction occurred in LB medium containing kanamycin (50 µg / mL). The cells were grown to an OD 600 between 0.4 and 0.8, induced with 0.1 mM IPTG (isopropyl thiogalactoside) and samples were taken 4 hours after induction. Cell extracts were prepared according to the protocol accompanying the Novagen BugBuster ™ reagent (Novagen, Madison, WI) (with benzonuclease and a mixture of Roche's complete protease inhibitor (Roche, Indianapolis, IN)). Very high levels of soluble protein with the expected molecular weight were obtained, as indicated by SDS-PAGE. For some reactions, the pET 30 gene product was purified using His-Bind cartridges according to the manufacturer's protocols (Novagen, Madison, WI). The eluent fractions were desalted on PD-10 columns (GE Healthcare, Piscataway, NJ) and eluted in 25-100 mM of potassium phosphate buffer, with a pH of 7.5.

Cell extracts were analyzed for D-aminotransferase activity by producing alanine from pyruvate and D-tryptophan using the following protocol. The 1 mL reactions were generally carried out in 100mM of potassium phosphate buffer (pH 7.5), 50 µM of pyridoxal phosphate, 25 mM of sodium pyruvate and 50 mM of D-tryptophan. Reactions were initiated by incorporation of cell-free extracts or purified enzymes and incubated for 15 minutes to 24 hours at 30 ° C with gentle shaking. Formic acid was added at the final concentration of two percent to stop the reaction and the precipitated protein was removed by centrifugation. Control reactions without added protein were also performed. Zero time points were also used as negative controls. Alanine was detected using an OPA derivatization as described in Example 1.

Example 8 (comparative example)

Comparison of total monatin production and isomeric distribution for aldolase activity polypeptides of SEQ ID NO: 8; SEQ ID NO: 4, SEQ ID NO: 12 and SEQ ID NO: 28 and ProA from C. testosteroni.

AT-103 transaminase (a broadly specific D-aminotransferase) was purchased from BioCatalytics (Pasadena, CA) and this enzyme or the recombinant enzyme produced in Example 7 was used in combined reactions with HMG aldolases to produce monatin from D tryptophan and pyruvate as described in published US patent application No. 20050282260. C. testosteroni aldolase ProA was used as a reference aldolase for comparative purposes and was prepared as described in patent applications not. 20040063175 and WO 03091396 A2 published. The aldolases tested were isolated and transformed as described in Example 4 above.

To produce sample amounts of each aldolase, 50 mL cultures were grown in LB medium containing ampicillin (100 µg / mL) at an OD 600 of approximately 0.5. Strains with SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 11 were induced with 100 µM of IPTG. The strain with SEQ ID NO: 27 was induced with 200 µg / L of anhydrotetracycline. Cells were cultured for 5 hours after induction and cell extracts were prepared according to the manufacturer's protocols (Novagen, Madison, WI, Bugbuster reagent). Inhibitors of benzonuclease and protease were added. Soluble proteins in cell extracts were separated on a Bio-Rad Laboratories Automated Electrophoresis-Experion Station and analyzed to verify their concentration and percentage using the Software version of Experion 1.1.98.0.

Incorporated for each 1 mL of reaction mixture: approximately 50 µg of aldolase (provided in cell extracts unless otherwise indicated), 4 mM of MgCl2, 50 mM of D-tryptophan, 0.5 mg of Daminotransferase of B purified sphaericus, 200mM sodium pyruvate, 100mM potassium phosphate buffer with a pH of 7.5 and 0.05mM PLP. The experiments were carried out in duplicate with negative controls to which no aldolase was incorporated. The samples were incubated for 1 hour, 2 hours and 24 hours (between 17 and 20 hours) at 30ºC with slight shaking. Small amounts of monatin (<0.5 ppm) are produced without aldolase in 24-hour reactions, given magnesium and phosphate catalyzed nonenzymatic reactions. These values were subtracted from the figures reflected below and the average results are shown. The only stereoisomers detected when monatin is produced using these procedures are the R, R and S, R stereoisomers. The percentage of R, R is listed below and was determined through the reversed phase LC peak area.

Table 3: Total monatin produced from D-tryptophan and% of R, R

% of (R, R) -monatin

89.7

79.0

94.8

93.8

100

98.65

100

97.35

86.45

63.1

The 18-hour sample of SEQ ID NO: 28 was also analyzed for stereoisomeric distribution by the FDAA derivatization procedure listed in Example 1, which gave a result of 94.9% (R, R) monatin and 5 0.1% (S, R) -monatin.

The same side-by-side experiments were performed using L-tryptophan as the initial substrate and combining the aldolases with L-aminotransferase of HexAspC broad specificity produced as described in published patent application No. 20050282260 and purified. Reactions should mainly generate (S, S) -monatin and (R, S) -monatin. Reactions were also supplemented with 10mM alpha-ketoglutarate as the amino acceptor for transamination of L-tryptophan. Again, the duplicate results are then averaged for total monatin (subtracting the basal levels without the presence of aldolase) and the percentage of (S, S) -monatin is reflected based on the peak area of reverse phase LC. In some cases, since aldolases are Specific and produce little monatin in total, the reversed phase estimates of the steroisomeric distribution are less accurate due to the asymmetry of the tryptophan peak that can co-elute with the (S, S) - peak. / (R, R) -monatin. The trends are still informative when comparing the R specificity of aldolases. The results of older

The analyzes using the FDAA derivatization procedure are reflected in parentheses for the different samples and are more accurate. Total monatin figures above approximately 400 ppm are greater than the linear range of the parameter scale used to quantify the results, making them qualitative results. C. testosteroni aldolase ProA generally produces between 95 and 100% (S, S) -monatin as reflected in published US Patent Application No. 2005028266.

10 Table 4: Total monatin produced from L-tryptophan and% of S, S

% of (S, S) monatin

78.9

78.15

95.65

87.6

93.55

75 (59.35)

55.05 (18.9)

27.25 (19.1)

92.5

92.15

It can be seen that the R specificity of the aldolase activity polypeptide of SEQ ID NO: 28 is relatively high relative to the reference ProA enzyme. This is also reflected in the low% (S, S) -monatin produced despite the high degree of specificity of the HexAspC aminotransferase for S-MP in these reactions. The numbers

15 monatin totals, when comparing (S, S) -monatin production with respect to (R, R) monatin production are not indicative of aldolase activity. D-aminotransferase is less active than HexAspC, especially at the MP concentrations that are present in these reactions.

For further comparison of the aldolase-active polypeptide of SEQ ID NO: 28 with the enzyme C. testosteroni ProA, various ratios of D-aminotransferase to aldolase were used in reactions that

20 started with D-tryptophan (no duplicate samples were made for these experiments). Reactions were carried out as explained above. For reactions in which the aldolase concentration was kept constant, approximately 50 µg was used. For reactions in which D-aminotransferase was kept constant, 0.5 mg was used. For the concentration of 2 and 10 mg / mL D-aminotransferase, a lyophilized enzyme was used. Duplicates were performed for the two highest concentrations of D-aminotransferase.

Table 5: Effect of D-aminotransferase concentration on the production of (R, R) -monatin

-

% of (R, R) monatin

100

97.1

-

% of (R, R) monatin

100

96.5

100

96.5

100

97.3

99.1

96.5

78.7

61.1

79.0

62.5

73.8

68.1

73.1

71.9

59.7

67.5

92.3

69.8

84.9

67.5

84.2

68.7

For monatin levels above 400 ppm, the results are not in the linear range of the standard curve and are approximate values only. The maximum amount of (R, R) -monatin produced, when properly diluted, was 1100 ppm. The FDAA stereoisomer analysis was performed for the aldolase activity polypeptide of SEQ ID NO: 28 with 10 mg / mL D-aminotransferase samples. At two hours, the

The sample contained 98.5% (R, R) -monatin. At 17 hours, the sample contained 95.9% (R, R) -monatin. The aldolase-active polypeptide of SEQ ID NO: 28 produced high percentages of (R, R) -monatin, even after long incubation periods and using large amounts of aminotransferase. If adequate Daminotransferase is provided, the aldolase-active polypeptide of SEQ ID NO: 28 produces as much monatin as C. testosteroni aldolase ProA indicating similar specific activity.

Table 6: Effect of aldolase concentration on the production of (R, R) -monatin

% of R, R monatin

100

97.4

97.3

95.7

93.3

78.2

73.2

64.1

63.0

62.5

61.5

88.1

74.7

78.2

67.8

70.1

63.9

When the aldolase concentration is varied, there is not much increase in total monatin. The percentage of R, R decreases with time and also with the concentration of aldolase, especially when there is not enough Daminotransferase.

In order to examine the specificity R of the tested aldolase, the experiments were carried out starting from L

Tryptophan and aminotransferase HexAspC, which was produced and purified as described in published US patent application No. 20050282260. HexAspc shows strong selectivity for transamination of S-MP versus R-MP, therefore, percentages above 50% (R, S) -monatin indicate a highly stereospecific aldolase. Ten mM of alpha-ketoglutarate was provided as an amino acceptor. However, at high concentrations, L-aminotransferase also uses pyruvate. In these reactions,

10 (S, S) -monatin and (R, S) -monatin generally only occur within the detection limits of the FDAA derivatization protocol.

Table 7: Effect of L-aminotransferase concentration on the production of (S, S) -monatin

Aldolasa

Daminotransferase concentration Weather Total monatin (approximate ppm) % of Monatina S, S

SEQ ID NO: 28

0.25 mg / ml 1 hour 13 33.8

SEQ ID NO: 28

0.25 mg / ml 24 hours 127 34.2

SEQ ID NO: 28

0.5 mg / ml 1 hour 31 30.9

SEQ ID NO: 28

0.5 mg / ml 24 hours 272 26.8

SEQ ID NO: 28

1 mg / ml 1 hour 3. 4 20.3

SEQ ID NO: 28

1 mg / ml 24 hours 385 23.5

ProA (purified) from C. testosteroni

0.25 mg / ml 1 hour 523 94.2

ProA (purified) from C. testosteroni

0.25 mg / ml 24 hours 1817 93.7

ProA (purified) from C. testosteroni

0.5 mg / ml 1 hour 602 91.8

ProA (purified) from C. testosteroni

0.5 mg / ml 24 hours 2122 89.9

ProA (purified) from C. testosteroni

1 mg / ml 1 hour 873 90.2

ProA (purified) from C. testosteroni

1 mg / ml 24 hours 1237 82.6

SEQ ID NO: 8

0.25 mg / ml 1 hour 339 86.3

SEQ ID NO: 8

0.25 mg / ml 24 hours 1499 88.0

SEQ ID NO: 8

0.5 mg / ml 1 hour 211 80.3

SEQ ID NO: 8

0.5 mg / ml 24 hours 1328 83.1

Aldolasa

Daminotransferase concentration Weather Total monatin (approximate ppm) % of Monatina S, S

SEQ ID NO: 8

1 mg / ml 1 hour 400 74.6

SEQ ID NO: 8

1 mg / ml 24 hours 1370 79.0

Table 8: Effect of aldolase concentration on the production of (S, S) -monatin

Aldolasa

Aldolase concentration Weather Total monatin (ppm) % of Monatina S, S

SEQ ID NO: 28

25! G / ml 1 hour eleven 25.1

SEQ ID NO: 28

25! G / ml 24 hours 112 20.0

SEQ ID NO: 28

50! G / ml 1 hour 18 31.8

SEQ ID NO: 28

50! G / ml 24 hours 160 27.0

SEQ ID NO: 28

100! G / ml 1 hour 33 33.2

SEQ ID NO: 28

100! G / ml 24 hours 238 41.4

ProA (purified) from C. testosteroni

25! G / ml  1 hour 305 86.4

ProA (purified) from C. testosteroni

25! G / ml  24 hours 1094 87.5

ProA (purified) from C. testosteroni

50! G / ml 1 hour 575 90.9

ProA (purified) from C. testosteroni

50! G / ml 24 hours 1449 89.9

ProA (purified) from C. testosteroni

100! G / ml 1 hour 817 93.6

ProA (purified) from C. testosteroni

100! G / ml 24 hours 1360 89.7

SEQ ID NO: 8

25! G / ml  1 hour 134 70.7

SEQ ID NO: 8

25! G / ml  24 hours 728 76.3

SEQ ID NO: 8

50! G / ml  1 hour 197 80.0

SEQ ID NO: 8

50! G / ml  24 hours 928 81.4

SEQ ID NO: 8

100! G / ml 1 hour 279 86.7

SEQ ID NO: 8

100! G / ml 24 hours 1383 86.8

For aldolases that are highly R-specific, such as the aldolase-active polypeptide of SEQ ID NO: 28, less total monatin is produced and the higher amount of aldolase increases total monatin (as well as% S, S). These aldolases produce less S-MP substrate, the preferred substrate for L-aminotransferase used. For enzymes that are less specific for R, such as ProA, the higher amount of aldolase does not significantly improve total monatin production or% (S, S) -monatin. Increasing the amount of Laminotransferase added decreases the percentage of (S, S) -monatin produced. Based on the above analysis, the polypeptide with aldolase activity of SEQ ID NO: 8 is between ProA and the polypeptide with aldolase activity of SEQ ID NO: 28 in terms of R-specificity, which coincides with the previous information where % R-MP is measured for the aldol stage only.

Subcloning of SEQ ID NO: 27

The following primers were used to PCR amplify the aldolase gene: 5'gaggagctcgagtcagacgtatttcagtcctttttc-3 '(SEQ ID NO: 385) and 5'-agaagacatatgatttatcagccggggac-3' (SEQ ID NO: 386). SEQ ID NO: 27 of the aldolase gene encodes the aldolase activity polypeptide of SEQ ID NO: 28. The resulting PCR product was digested with XhoI and NdeI to cut at the sites that had been integrated into the primers. The fragment was gel purified (QIAquick Gel Extraction Kit (Qiagen, Valencia, CA)) and ligated (using T4 DNA ligase) with pET28b which had been digested with XhoI and NdeI and gel purified. The ligation was transformed into chemically competent TOP10F 'cells. Colonies growing on the plates were monitored for inserts and several isolates with inserts were subjected to DNA sequence analysis (Agencourt, Beverly, MA).

Purification of the polypeptide with aldolase activity of SEQ ID NO: 28.

Confirmed aldolase clones were transformed into BL21 DE3 or BL21DE3 pLyss. The 24-hour cultures grown with the correct antibiotic were diluted in a clean medium (generally 1: 100) and grown to an OD600 of ~ 0.6 with aeration at 37 ° C. Cultures were induced with 1mM IPTG and changed at 30 ° C (with aeration) and incubation was continued overnight. Cells were cultured by centrifugation. The cell pellet was subjected to a freeze-thaw cycle to aid cell lysis. The cell pellet was lysed at BugBuster and Benzonasa (Novagen, Madison, WI) (according to the manufacturer's protocol). Cell debris was removed by centrifugation. The crude protein extract was applied to a HisBind column (Novagen, Madison, WI) that had been prepared according to the manufacturer's protocol. The column was washed and the protein was eluted according to the manufacturer's protocol. The purified protein was desalted with PD-10 columns (GE Healthcare, Piscataway, N.J.). The buffer used for the exchange consisted of 50 mM potassium phosphate with a pH of 7.5, 100 mM NaCI2, 4 mM MgCl2. The purified protein was concentrated with Amicon centrifugal concentrators (Millipore, Billerica, MA).

Example 9 (comparative example)

Comparison of total monatin production and isomeric distribution for polypeptides with aldolase activity of SEQ ID NO: 40, SEQ ID NO: 298, SEQ ID NO: 36, SEQ ID NO: 64, SEQ ID NO: 96, SEQ ID NO: 54, SEQ ID NO: 122, SEQ ID NO: 142, SEQ ID NO: 130, SEQ ID NO: 112, SEQ ID NO: 94, SEQ ID NO: 80 and SEQ ID NO: 28.

AT-103 transaminase (a broadly specific D-aminotransferase) was purchased from BioCatalytics (Pasadena, CA) and this enzyme or the recombinant enzyme produced in Example 7 was used in combined reactions with HMG aldolases to produce monatin from D-tryptophan and pyruvate as described in published US Patent Application No. 20050282260. The aldolase-active polypeptide of SEQ ID NO: 28 (labeled with histidine) was used as a reference aldolase for comparative purposes and was produced and purified as described at the end of Example 8. The other aldolases tested were isolated and transformed as described in Example 4 above.

To produce sample amounts of each aldolase, 25mL cultures were grown in LB medium containing ampicillin (100 µg / mL), at an OD600 of approximately 0.4. The strains were induced with 100 µM of IPTG. Cells were grown for 4 hours post-induction, and cell extracts were prepared according to the manufacturer's protocols (Novagen, Madison, WI, Bugbuster reagent) with benzonuclease. The soluble proteins in the cell extracts were separated on a Bio-Rad Laboratories Automatic Experion Electrophoresis Station (Bio-Rad, Hercules, CA) and analyzed to verify concentration and percentage using the Software version of Experion 1.1. 98.0.

Incorporated for each 1 mL of reaction mixture: approximately 50 µg of aldolase (provided in cell extracts unless otherwise indicated), 4 mM of MgCl2, 50 mg / mL of D-tryptophan, 2 mg of AT-103 (BioCatalytics, Pasadena, CA), 200mM Sodium Pyruvate, 100mM Potassium Phosphate Buffer with a pH of 7.5 and 0.05mM PLP. D-tryptophan is not soluble at this high concentration, but was used to ensure that the reactions were maintained in saturated amounts of D-tryptophan. The experiments were carried out in duplicate with negative controls where aldolase was not incorporated. The samples were incubated for 2 and 24 hours (between 17 and 20 hours) at 30 ° C with slight shaking. Small amounts of monatin are produced in 24 hours

no aldolase (~ 0.5 ppm) given magnesium and phosphate catalyzed nonenzymatic reactions. Typical values

for the% of (R, R) -monatin they are 50% for these samples. Negative control values were subtracted from

numbers reflected below and average results are shown. The only stereoisomers detected

5 when monatin is produced using these procedures are the R, R and S, R stereoisomers. The percentage of

R, R is listed below and was determined by the reversed phase LC peak area. The

same experiment after storing cell extracts and purified polypeptide with activity of

aldolase from SEQ ID NO: 28 for 2 months at -20ºC. In this case, 50 mM D-tryptophan was used as in the

Example 8. Twice the amount of aldolase was added with the exception of the polypeptide with aldolase activity of 10 SEQ ID NO: 28 for which 50 µg was used again. These results are reflected to the right of Table 9. The

FDAA derivatization results for the isomeric distribution are reflected in parentheses.

Table 9: Total monatin produced from D-tryptophan and% R, R

% of (R, R) -monatin

66.8

62.4

91.9 (91.6)

80.0

94.0

88.5

n / a

89.2

96.9

98.5

96.0

97.3

98.2 (98.8)

96.9

97.8 (99.1)

94.4

95.9

96.8

93.3

82.9

98.5 (99.6)

% of (R, R) -monatin

98.4

98.6

99.3

97.0

93.3 (96.4)

94.4

84.8

98.8

96.6

96.7 (99.6)

99.2

It can be seen that some enzymes are more stable to storage than other aldolases, based on the proportions of activity. A secondary product, most likely 4-hydroxy-4-methyl glutamate, can also be formed during these reactions. The mentioned enzymes were classified according to their specificity towards

Monatin production comparing its peak areas in relation to the by-product. The results were the polypeptide with aldolase activity of SEQ ID NO: 122, SEQ ID NO: 80, SEQ ID NO: 108, SEQ ID NO: 96, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO : 36, SEQ ID NO: 94, SEQ ID NO: 298, SEQ ID NO: 40, SEQ ID NO: 142, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: 62 .

Based on the initial experiments, the aldolase activity polypeptides of SEQ ID NO: 298, SEQ ID NO: 54

10 and SEQ ID NO: 42 appeared to be the most promising in terms of the level of activity and the% of (R, R) -monatin produced. Enzymes were subcloned into pET expression vectors with and without histidine tags.

Cloning of SEQ ID NO: 297, SEQ ID NO: 53 and SEQ ID NO: 41.

Primers used for cloning:

Table 10

SEQ ID NO: 297, SEQ ID NO: 53, and SEQ ID NO: 41 were PCR amplified and digested with the appropriate enzymes (NdeI and BamHI for the PCR products containing SEQ ID NO: 297 and SEQ ID NO : 53, NcoI and BamHI for the PCR products containing SEQ ID NO: 41) and gel purified (QIAquick Gel Extraction Kit (Qiagen, Valencia, CA)). SEQ ID NO: 297 and SEQ ID NO: 53 were individually ligated into pET28 which had been digested with NdeI and BamHI and gel purified. SEQ ID NO: 41 was ligated to pET30 which had been digested with NcoI and BamHI and gel purified. The ligature became TOP10. Colonies were monitored to verify inserts. Colonies isolated with an insert were subjected to DNA sequence analysis (Agencourt, Beverly, MA).

Aldolase Purification

Confirmed aldolase clones were transformed into BL21 DE3 or BL21 DE3 pLysS. The 24-hour cultures grown with the appropriate antibiotic were diluted in a clean medium (generally 1: 100) and reached an OD 600 of ~ 0.6 with aeration at 37 ° C. Cultures were subsequently induced with 1mM IPTG and changed at 30 ° C (with aeration) and incubation was continued for 24 hours. Cells were cultured by centrifugation. The cell pellet was basically subjected to a freeze-thaw cycle to aid cell lysis. The cell pellet was lysed at BugBuster and Benzonasa (Novagen, Madison, WI) (according to the manufacturer's protocol). Cell debris was removed by centrifugation. The crude protein extract was applied to a HisBind column (Novagen, Madison, WI) that had been prepared according to the manufacturer's protocol. The column was washed and the protein was eluted according to the manufacturer's protocol. The purified protein was desalted with PD 10 columns (GE Healthcare, Piscataway, N.J.). The buffer used for the exchange consisted of 50 mM potassium phosphate with a pH of 7.5, 100 mM NacI, and 4 mM MgCl2. The purified protein was concentrated with Amicon centrifugal concentrators (Millipore, Billerica, MA).

Purified aldolases test

The purified aldolases were assessed for their ability to produce (R, R) -monatin from Dtryptophan. Incorporated for each 1 mL of reaction mixture: approximately 50 µg of purified aldolase, 4 mM of MgCl2, 50 mM of D-tryptophan, 0.5 mg of purified B. sphaericus D-aminotransferase, 200 mM of pyruvate of sodium, 100mM potassium phosphate buffer with a pH of 7.5 and 0.05mM PLP. Samples were taken at 2 hours and at 24 hours. The results are reflected in Table 11 below.

Table 11: Total monatin produced from D-tryptophan and% R, R

Aldolasa (time point)

Total monatin (ppm) % of (R, R) -monatin (reverse phase LC peak area) % of (R, R) -monatin (FDAA derivatization)

SEQ ID NO: 298 (2 hours)

16.95 88.5 n / a

SEQ ID NO: 298 (overnight)

212 77.6 71

SEQ ID NO: 54 (2 hours)

12.05 96.7 n / a

SEQ ID NO: 54 (overnight)

161.85  93.0 91.1

SEQ ID NO: 42 (2 hours)

20.95 80.3 n / a

SEQ ID NO: 42 (overnight)

223.2 69.1 62.1

SEQ ID NO: 28 (2 hours)

14.25 95.8 n / a

SEQ ID NO: 28 (overnight)

176.6 93.4 92.3

The same experiments were performed using L-tryptophan as the initial substrate and combining aldolases with the broad specificity L-aminotransferase produced and purified as described in US Patent Application No. 20050282260 (0.5 mg protein purified). The results are

They are shown below in Table 12 for the total monatin production (subtracting the basic levels without the presence of aldolase) and the percentage of (S, S) -monatin is shown based on the peak area of reverse phase LC. Figures above 400 ppm are outside the linear range of the standard curve, and are approximate.

Table 12: Total monatin produced from L-tryptophan and% S, S

Aldolasa (time point)

Total monatin (ppm) % of (S, S) -monatin (reverse phase LC peak area) % of (S, S) -monatin (FDAA derivatization)

SEQ ID NO: 298 (1 hour)

186.6 64.0 n / a

SEQ ID NO: 298 (overnight)

197.5 64.3 67.6

SEQ ID NO: 54 (1 hour)

70.4 36.5 n / a

SEQ ID NO: 54 (overnight flush)

87.8 41.7 42.1

SEQ ID NO: 42 (1 hour)

401.1 80.9 n / a

SEQ ID NO: 42 (overnight)

507.5 82.9 85.8

SEQ ID NO: 28 (1 hour)

56.2 30.1 n / a

SEQ ID NO: 28 (overnight)

88.8 32.2 33.8

These data and the (R, R) -monatin data illustrate that for R-MP specificity, aldolase-active polypeptides have the following order: SEQ ID NO: 28, SEQ ID NO: 54, SEQ ID NO: 298, SEQ ID NO: 42.

Example 10 (comparative example)

Comparison of total monatin production and isomeric distribution for aldolase activity polypeptides of SEQ ID NO: 116, SEQ ID NO: 76, SEQ ID NO: 44, SEQ ID NO: 148, SEQ ID NO: 46, SEQ ID NO: 134, SEQ ID NO: 74, SEQ ID NO: 126, SEQ ID NO: 102, SEQ ID NO: 58, SEQ ID NO: 88, SEQ ID NO: 50, SEQ ID NO: 106, SEQ ID NO : 304, SEQ ID NO: 300 and SEQ ID NO: 28.

The recombinant enzyme produced in Example 7 was used in combined reactions with HMG aldolases to produce monatin from D-tryptophan and pyruvate as described in published US patent application No. 20050282260. The aldolase-active polypeptide of SEQ ID NO: 28 was used as a reference in these tests and had been purified as described in Example 8.

To produce sample amounts of each aldolase, 25 mL cultures were grown in LB medium containing ampicillin (100 µg / mL), until an OD600 of approximately 0.5 was reached. Cultures were induced with 1mM IPTG. Cells were changed at 30 ° C and developed within 24 hours. Cell extracts were prepared according to the manufacturer's protocols (Novagen, Madison, WI, Bugbuster reagent). Benzonuclease was also added. The soluble proteins in the cell extracts were separated on a Bio Rad Laboratories Experion Automatic Electrophoresis Station (Bio Rad, Hercules, CA) and analyzed for concentration and percentage using Software Experion version 1.1.98.0.

Incorporated for each 1 mL of reaction mixture: approximately 50 µg of aldolase (provided in cell extracts unless otherwise indicated), 4 mM of MgCl2, 50 mM of D-tryptophan, 0.5 mg of Daminotransferase of B purified sphaericus, 200mM sodium pyruvate, 100mM potassium phosphate buffer with a pH of 7.5 and 0.05mM PLP. Dithioteitrole (DTT) (final concentration of 2mM) was added to the samples described below. The experiments were carried out in duplicate. The samples were incubated for 2 hours and overnight (20 hours) at 30 ° C with slight shaking. The average results are shown below. The only stereoisomers detected when monatin was produced using these procedures are the R, R and S, R stereoisomers. The percentage of R, R is listed below and was determined by the peak area of reverse phase LC.

Table 13: Total monatin produced from D-tryptophan and% R, R

% of (R, R) -monatin

97

95

76

67

97

94

94

89

84

72

92

86

96

92

94

92

0

91

92

88

74

64

99

97

78

95

0

55

0

53

3. 4

% of (R, R) -monatin

36

55

40

99

97

Total monatin production numbers ranged from 1 ppm to over 200 ppm and the% R, R ranged from 0% to 99%. Since aminotransferase was the same for all aldolases, aldolase change can significantly affect both the amount of monatin produced and the stereoisomeric distribution of the

5 monatin produced. DTT (as in the samples below) seemed to increase the amount of total monatin produced.

The same experiments as above were performed using L-tryptophan as the starting substrate and combining the aldolases (provided as cell extract) with partially purified L-aminotransferase HexAspC (0.5mg HexAspC). The average results (of the duplicates) are reflected below in Table 14 for the total production of monatin (subtracting the basic levels without the presence of aldolase) and the% of (S, S) -monatin is reflected based on the reverse phase LC peak area. Figures above 400 ppm are outside the linear range of the standard curve and are approximate. The aldolase activity purified polypeptide of SEQ ID NO: 28 was used as a reference. The aldolase activity polypeptides of SEQ ID NO: 34 and SEQ ID NO: 300 (derived from plant) were used with and without 2mM DTT. Shannon and Marcus

15 (The Journal of Biological Chemistry 237; 3342-3347, 1962) used mercaptoethanol as a reducing agent in the original purification of aldolase from peanut HMG.

Table 14: Total monatin produced from L-tryptophan and% S, S

% of (S, S) -monatin

47.9

56.4

90.6

89.0

55.0

61.7

71.5

74.4

79.3

83.3

69.2

74.1

51.4

58.8

56.7

% of (S, S) -monatin

56.7

n / a

61.1

71.9

75.5

90.1

88.8

30.8

38.6

31.0

34.4

n / a

n / a

n / a

n / a

75.2

83

63.6

71.6

35.1

40.4

Example 11 (not claimed)

Effect of dithiothreitol (DTT) on monatin production

Several of the enzymes in Example 10 were chosen for further study. Plant-derived aldolases

5 showed improvement with the addition of DTT as a reducing agent. It was noted that aldolases derived from microbes from environmental samples also contain a high percentage of cysteine residues. Therefore, other experiments were performed to see if DTT also increased monatin production for non-plant aldolases.

For each 1 mL of reaction mixture, approximately 50 µg of aldolase (provided in

10 cell extracts unless otherwise noted), 4mM MgCl2, 50mM D-tryptophan, 2mg AT-103, 200mM Sodium Pyruvate, 100mM Potassium Phosphate Buffer with a pH of 7 , 5 and 0.05 mM PLP. Dithiothreitol (final concentration of 2mM) was added to the samples indicated below. The experiments were carried out in duplicate. The samples were incubated for 2 hours at 30 ° C with slight shaking. The average results are shown below for total monatin as determined by LC / MS / MS, with

15 the basic monatin production (without aldolase control) subtracted.

Table 15: Total monatin produced from D-tryptophan

Total monatin (ppm)

126

102

107

103

88

161

118

141

243

170

174

196

The non-aldolase control produced 10 ppm total monatin with and without DTT, indicating that DTT does not affect the overall reaction by reducing byproducts and does not affect D-aminotransferase activity. The aldolase activity polypeptides of SEQ ID NO: 46, SEQ ID NO: 58, SEQ ID NO: 28 demonstrated a benefit from DTT incorporation. The aldolase activity polypeptide of SEQ ID NO: 46 showed the greatest benefit, approximately 1.8-fold higher activity with 2mM DTT. Two aldolase-active polypeptides appear to have been inhibited by DTT (SEQ ID NO: 116 and SEQ ID NO: 50) while no effect was seen within the experimental error for the aldolase-active polypeptide of SEQ ID NO : 44. However, it is possible that for each aldolase used there is an optimal concentration of DTT to detect a benefit of providing the reducing agent.

The aldolase-active polypeptide of SEQ ID NO: 88 was chosen to study the effect of DTT concentration on monatin production. Combined reactions were performed as above. The results appear in Figure 15. The optimal concentration of DTT in this assay ranged from 2.5 to 5 mM for the amount of aldolase incorporated. The interesting thing is that if it did not include DTT, the amount of monatin produced was almost as high as 2.5 mM DTT, but incorporating suboptimal amounts of DTT (0.5-1mM) really seems to have an inhibitory effect as well as incorporation of a high amount of DTT (20mM).

Example 12

Comparison of total monatin production and isomeric distribution for aldolase activity polypeptides of SEQ ID NO: 278, SEQ ID NO: 162, SEQ ID NO: 276, SEQ ID NO: 178, SEQ ID NO: 202, SEQ ID NO: 166, SEQ ID NO: 218, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 244, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 264, SEQ ID NO : 268, SEQ ID NO: 272, SEQ ID NO: 184, SEQ ID NO: 282, SEQ ID NO: 186, SEQ ID NO: 192, SEQ ID NO: 200, SEQ ID NO: 280, SEQ ID NO: 284 , SEQ ID NO: 172, SEQ ID NO: 180, SEQ ID NO: 168, SEQ ID NO: 228, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 270, SEQ ID NO: 156 and SEQ ID NO: 28.

The recombinant enzyme produced in Example 7 was used in combined reactions with HMG aldolases to produce monatin from D-tryptophan and pyruvate as described in published US patent application No. 20050282260. The polypeptide with activity of Aldolase from SEQ ID NO: 28 was used as a reference in these assays and had been purified as described in Example 8.

To produce sample amounts of each aldolase, 25 mL cultures were grown in LB medium containing ampicillin (100 µg / mL), at an OD600 of approximately 0.5. Cultures were induced with 1mM IPTG. Cells were changed at 30 ° C and developed overnight. Cell extracts were prepared using a Bugbuster reagent according to the manufacturer's protocols (Novagen, Madison, WI). Benzonuclease was also incorporated. The soluble proteins in the cell extracts were separated in an Automatic Electrophoresis Station - Experion from Bio-Rad Laboratories (Bio-Rad, Hercules, CA) and analyzed to verify the concentration and the percentage using the software version of Experion 1.1. 98.0.

Approximately 200 µg of the aldolase activity polypeptides of SEQ ID NO: 278, SEQ ID NO: 162, SEQ ID NO: 276, SEQ ID NO: 178, SEQ ID NO were incorporated for each 1 mL of reaction mixture : 202, SEQ ID NO: 5 SEQ ID NO: 166, SEQ ID NO: 218, SEQ ID NO: 244, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 184, SEQ ID NO: 282, SEQ ID NO: 186, SEQ ID NO: 192 and SEQ ID NO: 200

or 50 µg of the aldolase activity polypeptide of SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 172, SEQ ID NO: 180, SEQ ID NO: 168, SEQ ID NO: 228, SEQ ID NO : 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 270 and SEQ ID NO: 156 (provided in cell extracts unless otherwise noted), 4mM MgCl2, 50mM 10 D- tryptophan, 0.5 mg purified B. sphaericus D-aminotransferase, 200 mM sodium pyruvate, 100 mM potassium phosphate buffer with a pH of 7.5 and 0.05 mM PLP. The experiments were carried out in duplicate. The samples were incubated for 2 hours and overnight (20 hours) at 30 ° C with slight shaking. The average results appear below. The only stereoisomers detected when monatin is produced using these procedures are the R, R, and S, R stereoisomers. The percentage of R, R is listed to

It was then continued and determined by the reversed phase LC peak area.

Table 16: Total monatin produced from D-tryptophan and% R, R

% of (R, R) -monatin

100

96

100

98

100

98

98

94

0

91

89

71

99

96

98

93

95

84

99

98

95

89

% of (R, R) -monatin

81

73

82

77

98

95

98

98

96

94

95

91

94

93

97

97

97

94

100

99

n / a

61

100

=

98

99

96

93

75

98

90

99

% of (R, R) -monatin

95

96

90

97

90

100

96

91

83

88

71

98

97

Total monatin production numbers ranged from undetectable to over 600 ppm and the% R, R ranged from 61% to 100%. Since aminotransferase was the same for all aldolases, aldolase change can have a significant impact on both the amount of monatin produced and the distribution

Stereoisomeric of the monatin produced.

The same experiments as above were performed using L-tryptophan as the initial substrate and combining aldolases (provided as cell extracts) with broad specificity L-aminotransferase HexAspC produced and purified as described in published U.S. patent application No 20050282260 (0.5 mg of purified protein). The results are reflected in Table 17 for the total production of monatin (subtracting the basic levels without the presence of aldolase) and the percentage of (S, S) monatin is reflected based on the peak area of reverse phase LC. Figures above 400 ppm are outside the linear range of the standard curve and are approximate. Table 12 shows the results for the reference Specific enzyme, the aldolase activity polypeptide of SEQ ID NO: 28 that was tested at the same time.

Table 17: Total monatin produced from L-tryptophan and% S, S

Aldolasa (time point)

Total monatin (ppm) Monatin S, S% (reverse phase LC peak area) % of (S, S) -monatin (FDAA derivatization)

SEQ ID NO: 278 (1 hour)

14.6 21.0 n / a

SEQ ID NO: 278 (overnight)

7905 24.6 n / a

SEQ ID NO: 162 (1 hour)

14 15.4 n / a

SEQ ID NO: 162 (overnight)

105.6 17.3 n / a

SEQ ID NO: 276 (1 hour)

35.8 10.2 n / a

SEQ ID NO: 276 (overnight)

67.8 9 15.1

Aldolasa (time point)

Total monatin (ppm) Monatin S, S% (reverse phase LC peak area) % of (S, S) -monatin (FDAA derivatization)

SEQ ID NO: 218 (1 hour)

11.7 20.3 n / a

SEQ ID NO: 218 (overnight)

49.9 17.7 22.0

SEQ ID NO: 244 (1 hour)

6.2 18.0 n / a

SEQ ID NO: 244 (overnight)

24.4 14.7 19.6

SEQ ID NO: 268 (1 hour)

6.3 24.1 n / a

SEQ ID NO: 268 (overnight)

61.2 23.3 29.2

SEQ ID NO: 272 (1 hour)

5.7 20.5 n / a

SEQ ID NO: 272 (overnight)

43.6 19.9 22.9

SEQ ID NO: 192 (1 hour)

6.8 19.0 n / a

SEQ ID NO: 192 (overnight)

56.4 20.0 24.4

SEQ ID NO: 172 (1 hour)

29.9 35.6 n / a

SEQ ID NO: 172 (overnight)

184.2 42.4 45.6

SEQ ID NO: 228 (1 hour)

59.6 23.9 n / a

SEQ ID NO: 228 (overnight)

182 35.6 38.0

Example 13 (comparative example)

Monatin production from indole-3-pyruvate using a D-aminotransferase

AT-103 transaminase was part of a transaminase library purchased from BioCatalytics (Pasadena, CA) and

5 The enzyme was tested for the production of monatin in combined reactions using the C. testosteroni ProA aldolase. Aldolase was prepared as described in Patent No. W0 03/091396 A2. AT-103 is a broadly specific Dtransaminase (E.C. 2.6.1.21) of the Bacillus species that requires a D-amino acid (such as a D-glutamate, D-aspartate, or D-alanine) as the amino acid donor. Enzymes and additional components / substrates were directly incorporated into the reaction buffer provided in the kit containing 100 mM of

10 potassium phosphate buffer with a pH of 7.5, 100 mM amino donor and 0.1 mM PLP. To one mL of reaction buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately 50 µg ProA provided in a cell extract, 1 µL 2M MgCl2, and 2 mg enzyme aminotransferase . Reactions were performed in duplicate. Reactions were incubated overnight at 30 ° C with slight shaking (100 rpm). The samples were filtered and subjected to reverse phase LC / MS / MS analysis as described in Example 1. The results

15 indicated that approximately 370 µg / mL monatin was produced using the enzyme AT-103. The results were subsequently analyzed to determine the proportions of S, R / R, S with respect to (R, R) - / (S, S) -monatin based on the peak areas of the two groups of stereoisomers that resolve. during chromatographic separation. Of the total monatin produced by AT-103, 69% was (R, R) - / (S, S) -monatin relative to the mixed isomers. This enzyme is homologous to the Bacillus subtilis DAT enzyme described in Patent No. WO

03/201396 A2 which is known to have broad specificity for D-amino acids. Chiral analysis was performed using the FDAA methodology described in Example 1, which verified that D-aminotransferase was primarily producing (R, R) -monatin and some (S, R) -monatin as expected. Other transamination experiments with (S, S) -monatin or (R, R) -monatin and α-ketoglutarate as substrates verified that the BioCatalytics enzyme was highly selective for carbon 4-D configuration as expected. In these experiments, no

Glutamate in reaction with (S, S) -monatin and α-ketoglutarate as substrates.

To decrease the amount of (S, S) -monatin or (R, S) -monatin produced as by-products in combined reactions with AT-103 (wide-range D-transaminase) and ProA aldolase, aldolase was purified using cartridges His-Bind according to the manufacturer's protocols (Novagen, Madison, WI.). The purified enzyme should preferably not contain wild-type L-aminotransferase activities that may be present in cell extracts (such as native E. coli TyrB or AspC activities). This His-Bind eluent was desalted to remove imidazole using PD-10 columns (G25 Sephadex, GE Healthcare, Piscataway, NJ) and eluted in 50mM Tris-Cl with a pH of 7. Experiments were performed in duplicate on a 1 mL volume and contained 100 mM Tris-Cl buffer, pH 7.8, 50 µg ProA aldolase, 4 mg indole-3-pyruvate, 1 or 2 mg Daminotransferase, 200 mM sodium pyruvate, 2mM MgCl2, 3mM potassium phosphate, 0.1mM PLP and 14.7mg D-glutamate. The tubes were incubated at 30 ° C with slight shaking. Two hour time points were taken and immediately frozen at -20 ° C. The pH was adjusted after two hours from 5 to 7 to 8 using NaOH and the assays were incubated overnight. The samples were filtered and analyzed to verify monatin as described in Example 1. The two-hour samples showed no detectable amounts of monatin, probably due to the low pH. The 24 hour samples contained about 190 ng / mL monatin when 1 mg D-aminotransferase was used and about 84% was (R, R) -monatin and 16% was (S, R) -monatin. When 2 mg D-aminotransferase was used, 540 ng / mL monatin was produced, about 71% was (R, R) -monatin.

Similar experiments were performed using a BioCatalytics aminotransferase buffer (BioCatalytics, Pasadena, CA), containing 100mM potassium phosphate with a pH of 7.5, 0.1mM PLP and 100mM Dglutamate. Solid indole-3-pyruvate and D-aminotransferase were added as above. ProA aldolase (50 µg), MgCl2, and 50 mM pyruvate were added from the concentrated solutions. The assays were treated as above, although a pH adjustment was not required in this case. A negative control was performed with the enzyme and the buffer provided by BioCatalytics, which did not contain monatin. The experimental results are reflected in Table 18.

Table 18: Monatin production from indole-3-pyruvate in phosphate buffer

Mg Daminotransferase

Time (hours) Total monatin (ng / mL) % of R, R

0

2  0 n / a

one

2 6780 Undetermined

2

2

13170 55%

0

16  0 n / a

one

16 15000 Undetermined

2

16 28930 51%

Monatin production in a phosphate buffer is clearly higher than in Tris buffered systems.

To compare the activities of the cloned B. subtilis DAT from WO 03/091396 A2 with the enzyme from BioCatalytics (AT-103) (BioCatalytics, Pasadena, CA), additional tests were performed. The B. subsubtilis dat gene was also subcloned into pET30 to remove the Histidine 6 label. Labeled and unlabeled enzymes were produced in BL21 (DE3) as described in WO 03/091396 A2. Cell extracts were made and total protein assays were performed to estimate protein concentration as previously described. Duplicate one mL reactions were performed containing: 500 µg D-aminotransferase, 50 µg ProA aldolase, 100 mM potassium phosphate with a pH of 7.5, 3 mM MgCl2, 4 mg indole-3 -pyruvate, 200 mM sodium pyruvate, 7.35 mg (50 mM) D-glutamate and 0.1 mM PLP. The samples were incubated at 30 ° C for 1, 2 and 24 hours and filtered for analysis by LC / MS / MS. The samples only contained monatin S, R and R, R stereoisomers as determined by the FDAA derivatization protocol described in Example 1. The results are summarized in Table 19 below. The% R, R was determined by peak areas that were separated by reverse phase chromatography.

Table 19: Comparison of D-aminotransferase enzymes

% of (R, R) -monatin

-

undetermined

undetermined

% of (R, R) -monatin

undetermined

-

80-90%

080%

92.5%

-

31

33

66

Removal of the Histidine 6 tag appears to have improved B. subtilis D-aminotransferase activity. However, the BioCatalytics D-aminotransferase homolog clearly had the highest activity. The BioCatalytics D-aminotransferase homolog also demonstrated higher substrate specificity for the R-monatin precursor. The longer incubation periods seem to reduce the enantiomeric excess of R, R, monatin that is produced.

Since Bacillus D-aminotransferase enzymes have a preference for pyruvate as an amino acceptor and for D-alanine as an amino donor, it was hoped that D-alanine could be used as the amino donor for the conversion of MP to monatin with better or similar results. Duplicate reactions of one mL were performed containing: 500 µg of D-aminotransferase, 50 µg of purified ProA aldolase, 100 mM of potassium phosphate with a pH of 7.5, 3 mM of MgCl2, 4 mg of indole. 3-pyruvate, 100 mM sodium pyruvate, 25 mM Dglutamate or D-alanine and 0.1 mM PLP. The samples were incubated for 2 hours and treated as before prior to analysis. When D-alanine was used as the amino donor, monatin levels were produced slightly higher than expected (23 vs. 21 ppm). Also, it is hoped that high concentrations of pyruvate can inhibit the transamination step, so administering pyruvate doses in smaller amounts over time can improve the overall proportion of monatin production. It can be seen from the above data that even though half the pyruvate was used in this case, relative to the table above, a significantly higher amount of monatin was produced. AT-103 is an example of an enzyme with limited activity for S-MP. Despite the fact that the ProA aldolase used in this study generates more than 90-95% of S-MP, AT-103 generates up to 92% of (R, R) -monatin.

Example 14 (comparative example)

Production of (R, R) -monatin from D-tryptophan

Approximately 60 µg of C. testosteroni ProA aldolase (provided in cell extracts as described in WO 03/091396), 4 mM MgCl2, 50 mM D-tryptophan were incorporated per 1 mL of reaction mixture , 0.5mg D-aminotransferase (AT-103) from BioCatalytics (BioCatalytics, Pasadena, CA), 100mM Sodium Pyruvate, 100mM Potassium Phosphate Buffer with a pH of 7.5 or 100mM Buffer sodium acetate with a pH of 8, 0.05 mM PLP, 3 mM potassium phosphate (for acetate reactions only), and 10 mM a-ketoglutarate. The experiments were carried out in duplicate with negative controls where aldolase was not added. The samples were incubated overnight (20 hours) at 30 ° C with slight shaking. The actual pH of the sodium acetate samples was approximately 5, while the final pH for the phosphate buffered samples was approximately 7. None of the aldolases appeared to have significant activity at a pH of 5; the sample containing ProA aldolase was just above the negative control but was probably not above the experimental error. In potassium phosphate, ProA aldolase produced 73.4 ppm monatin with an R, R: S, R ratio of 1.7: 1 (~ 63% R, R from D-tryptophan).

Since Bacillus D-aminotransferase enzymes have a preference for pyruvate as an amino acceptor and for D-alanine as an amino donor, the incorporation of alpha-ketoglutarate was expected to be unnecessary when R, R or (S was produced , R) -monatin from D-tryptophan. The above experiment was repeated (in 100 mM potassium phosphate buffer) using purified ProA aldolase (50-60 µg) and an incubation time of 2.5 hours. Duplicate experiments were performed with and without alpha-ketoglutarate. With the incorporation of 10 mM alpha-ketoglutarate, 56.1 ppm monatin was formed from D-tryptophan (79.5% R, R, 20.5% S, R). Without alpha-ketoglutarate, 102.5 ppm monatin was formed (79% R, R, 21% S, R).

Example 15 (comparative example)

Tryptophan racemase

(R, R) -monatin has been produced using D-aminotransferase and an aldolase when D-tryptophan was used as the starting material (Example 14). Notwithstanding the foregoing, L-tryptophan may be a preferred starting material for several reasons. For example, L-tryptophan may be cheaper and more available than Dipryptophan. The present application describes various procedures for obtaining an active tryptophan racemase. The yields of (R, R) -monatin are improved using an R-specific aldolase, that is, an aldolase that preferentially or selectively produces R-MP. Figure 3 illustrates a procedure for producing stereoisomer-enriched (R, R) -monatin from L-tryptophan using tryptophan racemase, a D-aminotransferase, and an R-specific aldolase.

A selection for a tryptophan racemase is created by constructing a strain that would require an active racemase for growth. An auxotrophic tryptophan needs a source of L-tryptophan when it grows in minimal medium. Supplementing the medium with D-tryptophan is one way to select for a racemase that converts D-tryptophan to L-tryptophan. Auxotrophic tryptophans were tested for growth in minimal medium supplemented with D-tryptophan. The strains, CAG18455 and CAG18579 from the Coli Genetic Stock Center and NRRL12264 (also lipA-, lysogenized and cured ADE3 from their plasmid) did not grow when supplemented with D-tryptophan but grew when supplemented with L-tryptophan. E. coli can be used as a host organism but other host organisms such as yeast, other bacteria, or other eukaryotic organisms can also be used. An auxotrophic tryptophan (especially NRRL12264 (also called lipA, lysogenized ADE3 and cured from its plasmid)) will grow into D-tryptophan when it has been transformed with a D-aminotransferase. This confirms the ability of E. coli to transport D-tryptophan in the cell.

Saicher and Lingens described the presence of a tryptophan racemase in Pseudomonas aeruginosa (ATCC 15926). Tryptophan racemase has also been described in several plants, including tobacco, beets, tomatoes, and wheat, and the enzyme appears to be induced by stress conditions or osmotic drought. Tryptophan racemase may play a role in Sclerochiton ilicifolius in the native monatin production pathway. To isolate this racemase activity, an expression library is constructed from ATCC15926 (or another organism with tryptophan racemase activity) and the library is transformed into the auxotrophic tryptophan. A strain is selected for cultivation using D-tryptophan as the source of tryptophan. A similar procedure is also used to monitor many strains with known racemases for a racemase with D-tryptophan activity. Examples include: alanine, serine, and glutamate racemases (T. Yoshimura and N. Esaki, "Amino Acid Racemases: Functions and Mechanisms". Journal of Bioscience and Bioengineering, Vol. 96, No. 2, 103-109, 2003). Alanine racemase is PLP dependent and has been cloned from Salmonella typhimurium (dadB gene). Other sources include Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Schizosaccaroyces pombe, and Bacillus cereus. A basidiomycete fungus, Lentinus edodes, also contains a highly active alanine racemase. Serine racemase is also PLP dependent and is found in eukaryotes (such as the silkworm, rat brain, mouse brain cDNA) as well as bacteria (Enterococcus gallinarum). Glutamate racemase is PLP independent and has been cloned from Pediococcus pentosaceus, Bacillus pumilus, Lactobacillus fermenti, Lactobacillus brevis, E. coli, Aquifex pyrophilus, and Bacillus subtilis. Glutamate racemase is very specific and therefore even amino acids aspartate, asparagine, and glutamine of similar structure are not substrates for the enzyme. Aspartate racemases also exist and are independent of PLP. These enzymes are found in strains of Lactobacilli, Streptococcus, and some archaeas such as the Desulfurococcus and Thermococcus strains. The bivalve mollusk Scapharca brouhtonii also contains an aspartate racemase. Other racemases found in the library include amino acid racemase (EC 5.1.1.10) from Anabaena sp. and Pseudomonas striata, proline racemase, multifunctional phenylalanine racemase. Related epimerases or racemases are also tested. Potential racemases are tested to ensure they are not D-tryptophan aminotransferases. This monitoring is done by sequence analysis and / or an enzyme assay.

Enzymes that pass the test as a racemase are monitored to verify monatin activity as described in Example 17. Ideally, an enzyme is obtained that is highly specific for tryptophan and has little or no racemase activity in monatin.

A tryptophan racemase can also evolve and / or improve (by mutagenesis or recombinant engineering) from an existing racemase, transaminase, or epimerase. Also, since crystal structures for alanine aminotransferases are known, they can be used as the basis for rational structure-based mutagenesis. The process described above is used as an initial screen for tryptophan racemase activity and as a screen for better activity.

Tryptophan racemase libraries

Library construction:

Burkholderia pyrrocina (ATCC15958) and Pseudomonas chlororaphis (ATCC15926) were obtained from the American Type Culture Collection. They were developed as suggested by ATCC and genomic DNA was prepared according to the Mekalanos JJ procedure. (Duplication and Amplification of Toxic Genes in Vibrio cholerae. Cell. 1983.

35: 253-63). Genomic DNA was partially digested with the restriction enzyme Sau3AI. Fragments 1-3 Kbp were gel purified using a Qiaquick Gel Extraction Kit from Qiagen (Qiagen, Valencia, CA). The purified DNA was ligated into pTrc99a (Amersham, Piscataway, NJ) which had been digested with BamHI and purified as indicated above. Ligation was performed at room temperature with a 24 hour incubation using a 3: 1 insert-vector molar ratio. The ligated library was transformed into chemically competent TOP10F cells (Invitrogen, Carlsbad, CA) and plated on LB medium with 100 µg / ml ampicillin. After 24 hour incubation of the transformation plates, colonies were removed from the plates, washed with liquid LB medium, and the appropriately sized cell pellet was prepared using a QIAquick miniprep kit from Qiagen (Qiagen, Valencia, CA ). About 30,000 colonies were assembled and prepared.

The pooled plasmid was transformed into CAG18455 (trpC83: Tn10, rph-1) or CAG18579 (trpC: Tn10kan, rph-1). Both strains are tryptophan auxotrophs so they will not grow in a minimal M9 medium (Difco) unless the medium is complemented with tryptophan. Transformants were plated on M9 minimal medium supplemented with D-tryptophan. This selects for a strain that can convert D-tryptophan to L-tryptophan.

Before transformation of the library, the strains were tested for growth in minimal medium with L-tryptophan or D-tryptophan. The strains were tested for growth in minimal medium supplemented with D-tryptophan and no growth was observed. Both strains grew in identical media supplemented with L-tryptophan instead of D-tryptophan. Also, a derivative of NRRL12264 (the strain used had been cured from the tryptophan operon plasmid, lysogenized with ADE3, and removed for lipA, along with other chromosomally encoded mutations (serB, �trpED, tnaA2, aroP) (this strain was unable to grow in minimal medium supplemented with D-tryptophan but grew in identical medium supplemented with L-tryptophan instead of D-tryptophan) was transformed with a Bacillus subtilis specific D-aminotransferase (WO 03/091396). -aminotransferase was driven by the T7 promoter The transformed strain was able to grow in M9 minimal medium supplemented with D-tryptophan.

Colonies growing on D-tryptophan medium are monitored. The plasmid is isolated and transformed back into a mother strain (CAG18455 or CAG 18579) to confirm that growth in D-tryptophan medium depends on the plasmid and not on a host mutation. The nucleotide sequences of the plasmids that complement the auxotrophic tryptophan are analyzed. Clones considered to have a tryptophan racemase gene are subsequently analyzed.

Tryptophan racemase from other tissue sources is isolated in a similar way. There are collection reports of tryptophan racemase activity both in cells in tobacco tissue culture (Nicotiana tabacum L. var. Wisconsin 38) (Miura, GA and Mills, SE The conversion of D-tryptophan to L-tryptophan in cell cultures of tobacco. Plant Physiol. 1971 47: 483-487) as in crude protein extracts of wheat (Triticum aestivum) (Rekoslavskaya, NI, Yur'eve, OV, Shibanova, LA, and Salyaev, RK Synthesis and physiological function of Dtryptophan during wheat germination. Russian Journal of Plant Physiology 1997. 44: 196-203) A cDNA expression library is created from tissue, as described in the collection, and the expression library is used to transform an auxotrophic tryptophan as previously described.

Tryptophan racemase assay

Clones that are identified as those that potentially have tryptophan racemase are transformed into a strain of E. coli commonly used for the expression of recombinant proteins, such as BL21. The cells are grown in the LB broth at a 600nm optical density of 0.4-0.6. The promoter that drives the expression of the racemase is induced with IPTG (0.1 mM of final concentration). After induction, cells can express the protein for 1 to 3 hours at 37 ° C (with aeration). Cells are cultured and smoothed by plunger filter, sonication, or by chemical means (such as BugBuster (Novagen, Madison, WI)). The lysed cells are spun to remove cell debris. The clarified extract was used directly in the trials.

Variable amounts of extract are added in a solution such that the final concentration is 50mM potassium phosphate (pH 7.0) and 2mM L-tryptophan. Pyridoxal-5'-phosphate is added at a final concentration of 10 µM. The samples are incubated and subsequently analyzed by LC / MS. The presence of a D-tryptophan peak when only L-tryptophan is used as a substrate indicates a positive result. Dryptoptophan concentration should increase with increasing time until equilibrium is reached and the rate should also increase with protein concentration until the enzyme concentration is high enough that it is no longer saturated with the substrate. D-tryptophan can also be converted to L-tryptophan as indicated above.

A complementary gene can be encoded for a D-aminotransferase. (A "complementary gene" is a gene that, when expressed, cancels out a mutation in an organism. For example, if an organism has a zero mutation in one of the genes necessary for the synthesis of tryptophan by the cell, a complementary gene might be one that, when expressed, allows the strain to grow in minimal medium (ie, without tryptophan.) This reaction requires an alpha-keto acid as an α-ketoglutarate, oxalacetate, or pyruvate as an amino acceptor. are present in a cell extract (in small amounts) These compounds can be removed using a PD-10 desalination column (GE Healthcare, Piscataway, NJ) and the assay can still be run in crude extract. Tryptophan racemase activity is purified using a Conventional column chromatography Finally, the open reading frame identified as a potential tryptophan racemase is cloned into an expression vector with an affinity tag. The potential is subsequently purified by affinity chromatography. In both cases, the purified protein is used in enzyme assays basically as described above.

Reverse Genetic Engineering of Tryptophan Racemase

Tryptophan racemase can be purified from plant or microbial sources by traditional protein purification techniques including ammonium sulfate fractionation and conventional column chromatography. Once the protein has been purified such that a sample can be isolated on a 2-D gel, conventional peptide microsequence or amino acid sequence techniques of the Edman type are used (see golgi.harvard.edu/microchem/ for descriptions of protocols and equipment used for this type of work). However, in some cases, the body's genome sequence cannot be used as a source of the protein for protein purification because that sequence has not yet been determined. In such a situation, the first set of degenerate primers can be designed based on an available sequence of the closest known relative of the protein source. The degenerate PCR and genome are subsequently developed according to established protocols to isolate the coding sequence of tryptophan racemase.

Monatin tryptophan racemase production

Incorporated for each 1 mL of reaction mixture: approximately 60 µg of C. testosteroni ProA aldolase (provided in cell extracts as indicated in patent WO 03/091396 A2), 100 µL / mL of tryptophan cell extract racemase or 1 mg / mL tryptophan purified racemase, 4 mM MgCl2, 50 mM L-tryptophan, 0.5 mg D-aminotransferase from BioCatalytics (AT-103) (BioCatalytics, Pasadena, CA), 100 mM pyruvate sodium, 100mM potassium phosphate buffer with a pH of 7.5, 0.05mM PLP and 10mM a-ketoglutarate. Since pyruvate is an acceptable amino acceptor for broad specificity D-aminotransferase, acetoglutarate is optional. The experiments are carried out in duplicate with negative controls where aldolase or tryptophan racemase is not incorporated. The samples are incubated for 1 hour or overnight (20 hours) at 30 ° C with slight shaking.

Tryptophan racemase is tested to verify monatin activity. An assay similar to that of Example 17 was used with monatin as a substrate and compared with the activity of the enzyme in tryptophan. The ideal enzyme has activity in tryptophan but has little or no activity in other amino acids, especially glutamate and monatin. If the enzyme has significant monatin activity, the enzyme can be mutagenized to decrease monatin and / or glutamate activity while keeping tryptophan activity intact or at a level high enough for the enzyme to be useful in monatin production. Techniques that can be used for mutagenesis include, but are not limited to, error-prone PCR, site-directed mutagenesis, modeling to identify targets for site-directed mutagenesis (sites that can be included in substrate binding) , passage through mutagenic strains and DNA transposition.

Mutagenized racemases can be monitored to verify tryptophan activity using a plaque assay as described above. Clones that retain tryptophan activity are monitored for loss of activity in monatin.

Example 16 (not claimed)

HEXAspC site-directed mutagenesis

Experimental summary

The E. coli AspC hexamutant (HEXaspC) was found to have better activity compared to AspC for the production of (S, S) -monatin as described in Example 6 of WO 03/091396 A2. The HEX (accession number: / AHFA gi: 127190) contains the following AspC mutations (numbering for E. coli): V35L, K37Y, T43I, N64L, T104S and N285S. Based on structural analysis and literature reports (S. Rothman and J. Kirsch,

J. Mol. Biol. (2003), 327, 593-608; S. Rothman et al., Protein Science (2004), 13: 763-772), 5 additional mutants were created that were expected to increase the kinetic activity of the substrates used in the monatin production pathway: L-tryptophan, S-MP or both. Two of the mutants increased transamination rates for tryptophan and (S, S) -monatin. Two of the mutants showed increased stereoselectivity for (S, S) -monatin formation while one was less stereoselective. Based on this, the broad specificity Daminotransferase of Bacillus sp. having similar mutations is useful as Daminotransferase in the (R, R) -monatin pathways shown in Figure 3 and described in Example 15. One of the mutants (HEXaspCP9T / R122G) exhibited superior activity for transamination of L -tryptophan, but the activity in the production of (S, S) -monatin or in the transamination of (S, S) -monatin decreased significantly. Therefore, this enzyme is expected to be useful in the first stage of the pathways of

production of the (R, R) -monatin shown in Figures 1, 2, 4, 5, 6, 7 and 8 and described in Examples 18 and 19. In general, aminotransferase exhibiting an activity similar to that of AspC on L-tryptophan and limited activity on R-MP and S-MP would be useful for the processes described in Figures 1, 2, 4, 5, 6, 7 and 8.

Procedures and materials

The HEX gene cloned into pUC19 was provided by Professor J. F. Kirsch (Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, California 94720-3206) and used as the template for cloning the gene into pET23a. See James J. Onuffer and Jack F. Kirsch, Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis, Protein Science, 4: 1750-1757 (1995). See also NCBI accession number 1AHF_A GI: 1127190 (HEX amino acid sequence). The following primers were designed to clone the HEX gene into the pET23a vector (Novagen, Madison, WI.):

HEXaspC primers:

''

Terminal C: 5'-ATAACCGGATCCTTACAGCACTGCCACAATCG-3 '(SEQ ID NO: 394)

The following PCR protocol was used for gene amplification: In a 100 µL reaction, 50 ng of DNA template was added, 1.0 µM of each primer, 0.2 mM of each dNTP, 1 U Pfu Turbo Polymerase (Stratagene, La Jolla, CA), and Pfu template cloned once. The thermocycler program used a warm start at 94 ° C for 5 minutes; followed by 25 cycles of a denaturation stage at 94ºC (30 seconds), an annealing stage at 55ºC (1 minute) and an extension stage at 72ºC (2 minutes) and finally a finishing stage at 72ºC (7 minutes). Standard molecular biology techniques were used to clone the PCR product into pET23a using the NdeI and BamHI restriction sites.

The tryptophan residue at position 130 is thought to be important in binding interactions with the pyridoxyl ring, but also appears to be a source of steric hindrance to the S-monatin precursor (MP) substrate, based on observations of protein modeling. Therefore, an amino acid with a smaller hydrophobic side chain (phenylalanine) was used to replace tryptophan. The rest of the mutations were based on kinetic data in the literature, although new combinations of desirable mutations were created. All mutations to HEXaspC, with the exception of W130F, were made using the Stratagene Multi-Change Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The W130F mutation was performed using the QuikChange kit from Stratagene (Stratagene, La Jolla, CA) in accordance with the manufacturer's instructions with the sole exception that the extension temperature for the PCR reaction decreased to 66 ° C. The primers for the multi-change kit (multi-change) were designed using the QuikChange multi-kit primer design tool at <www.stratagene.com>, except for the W130F single mutation primers.

Primer sequences are listed below in Table 20.

Table 20

Primer

Sequence (5 ’to 3’)

aspCW130F_backward

CGCTCTTATGGTTCGGTTTGCTTGGGTTGCTCACCC (SEQ ID NO: 395)

aspCW130F_forward

GGGTGAGCAACCCAAGCTTTCCGAACCATAAGAGCG (SEQ ID NO: 396)

R122G-1a

 CAAAAAATACCAGCGTTAAGGGAGTGTGGGTGAGCAACC (SEQ ID NO: 397)

P9T_4a

 CATTACCGCCGCTACTGCCGACCCGATTC (SEQ ID NO: 398)

I68V-1a

 CACCAAAAATTACCTCGGCGTAGACGGCATCCCTGAATT (SEQ ID NO: 399)

T156Aa

 TGATGCGGAAAATCACGCTCTTGACTTCGATGCAC (SEQ ID NO: 400)

aDenotes a primer that was modified by 5 'phosphorylation

Expression of HEXaspC mutant genes and analysis of enzyme activity

Liquid cultures (5 mL) of the Novagen Overnight Express ™ Autoinduction System 2 (Catalog # 71366-3; Solutions 1-6) (Novagen, Madison, WI) were inoculated from the fresh plates or from the frozen glycerol stocks of the following strains:

E. coli BL21 (DE3) :: HEXaspCpET23a

E. coli BL21 (DE3) :: HEXaspCW130FpET23a

E. coli BL21 (DE3) :: HEXaspCT156ApET23a

E. coli BL21 (DE3) :: HEXaspCP9T / T156ApET23a

E. coli BL21 (DE3) :: HEXaspCP9T / R122GpET23a

E. coli BL21 (DE3) :: HEXaspCR122G / T156ApET23a.

The cultures were incubated at 37 ° C at 230 rpm for 6 to 8 hours. The OD600 of each culture was determined and the volume of culture necessary to obtain an OD600 of 0.03 to 0.05 in 25 mL was calculated. The calculated volumes of each liquid culture were transferred to flasks containing 25 mL of the same medium. The Overnight Express ™ Autoinduction System 2 is a comprehensive, chemically defined medium for high level expression with IPTG-induced expression systems that use lactose as an induction agent and do not require control of cell growth. Overnight Express cultures were incubated at 30 with shaking at 230 rpm for 18 hours. Cells were harvested by centrifugation and washed once with cold 50 mM MOPS, pH 7.0. Cells were then lysed using Bugbuster ™ (Primary Amine Free) Extraction Reagent (Novagen Catalog # 70923-3) (Novagen, Madison, WI) containing 1 µL / mL benzase nuclease (Novagen Catalog # 70746-3) (Novagen, Madison, WI), 5 µL / mL Group II Protease Inhibitor Cocktail (Novagen Catalog # 539132) (Novagen, Madison, WI) and 0.33 µL / 10 mL r- Lysozyme (Novagen Catalog # 71110-3) (Novagen, Madison, WI) according to Novagen's recommended protocol. After incubation at 25 ° C for 15 minutes with gentle shaking, the cell debris from each suspension was pelleted by centrifugation at

21,000 g for 15 minutes at 4ºC. The supernatant was carefully decanted and analyzed as the cell-free extract. The inclusion body fractions were isolated by suspending the cell debris fractions in 30% Bugbuster ™ Extraction Reagent (primary amine free), centrifugation at 21,000 × g for 10 minutes; suspension of the centrifuged granules in 10% Bugbuster ™ extraction reagent (primary amine free), and centrifugation again to isolate the washed granules. Cell-free extracts and inclusion body fractions were analyzed to verify protein expression by SDS-PAGE on 4 to 15% gradient gels (BioRad # 161-1104, Hercules, CA). For cell extract samples, twenty micrograms of soluble protein was loaded into each gel pathway (pre-mixed with 1 × protein loading buffer and heated at 95 ° C for 5 minutes). The inclusion body fractions were dissolved in 1 × protein loading buffer (0.2 mL), heated for 10 minutes at 95 ° C, and 5 µL of each solution was loaded per gel route. The amount of each HEX mutant compared to the total soluble protein loaded in each pathway was calculated by band intensity analysis using the Labworks BioImaging 1D gel tool (UVP, Inc. Upland, CA) and reported to continuation:

Table 21

Shows

HEXaspC protein / total soluble protein

E. coli BL21 (DE3) :: HEXaspCP9T / T156ApET23a CFE

0.310

E. coli BL21 (DE3) :: HEXaspCP9T / R122ApET23a CFE

0.145

E. coli BL21 (DE3) :: HEXaspCpET23a CFE

0.172

E. coli BL21 (DE3) :: HEXaspCR122A / T156ApET23a CFE

0.174

E. coli BL21 (DE3) :: HEXaspCW130FpET23a CFE

0.114

Shows

HEXaspC protein / soluble protein  total

E. coli BL21 (DE3) :: HEXaspCT156ApET23a CFE

0.120

Analysis of the gels showed that the HEXaspCR122A / T156A mutant was the only protein found in substantial amounts as inclusion bodies. The HEXaspCP9T / T156A protein provided the highest level of expression, approximately 90% better than the HEXaspC protein. In contrast, W130F, T156A, and P9T / R122G proteins were expressed at lower concentrations than HEXaspC.

The activity of the HEXaspC mutant proteins for the production of S, S-monatin was measured using the following reaction conditions: Each 1 mL of reaction contained 50 mM TAPS, pH 8.2, 4 mM MgCl2, 3 mM sodium phosphate, pH 8.0, 200 mM sodium pyruvate (pH adjusted to 8), 5 mM a-ketoglutarate (pH adjusted to 8), 50 mM tryptophan, 0.05 mM pyridoxal 3- phosphate, 50 µg / mL ProA aldolase (added as a cell-free extract) and varying concentrations (approximately 50 and 500 µg / mL) of aminotransferase (added as a cell-free extract). Deaerated water was used to prepare the stock solutions and to adjust the volume of the reaction mixtures to 1.0 mL. Pyroxidal phosphate was added just prior to the addition of the enzymes. Reaction tubes were incubated at 30 ° C with gentle shaking for 4 hours. Samples (0.01 mL) were removed at 1, 2, and 4 hours after addition of the enzymes, filtered, and analyzed by LC / MS / MS, as described in Example 1. Monatin production it was normalized based on the amount of aminotransferase present in the reactions. Under the conditions of these assays, HEXaspC and HEXaspCT156A produced the highest amount of monatin per mg aminotransferase while P9T / R122G protein produced the lowest, followed by HEXaspCW130F. The HEXaspCW130F and P9T / R122G enzymes showed the highest stereoselectivity for S-MP (greater than 98% S, S-monatin), even when high concentrations of enzymes (greater than 300 µg / mL) were used. The percentage of S, S-monatin product decreased to less than 90% in the enzymatic reactions containing the enzyme P9T / T156A in a high concentration. The other mutants showed very similar product stereoselectivity to the original HEXaspC mutant (approximately 95% S, S-monatin). Analysis of the reaction product containing the HEXaspC enzyme using the FDAA derivatization reagent described in Example 1 showed that the second stereoisomer formed was R, S-monatin.

Assay of tryptophan and monatin aminotransferase activity

Mutants were tested to verify transamination activity by using (S, S) -monatin and L-tryptophan as substrates. Aminotransferase activity was measured by monitoring the formation of the reaction co-product, glutamate, by HPLC with post-column derivatization with OPA as described in Example 1. The reaction mixture contained, in 1.0 mL, 100 mM HEPPS buffer, pH 8.0, 20 mM alpha-ketoglutarate, 0.08 mM pyridoxal phosphate, 25 mM tryptophan or (S, S) -monatin and enzyme (provided as 2.5 mg protein from cell extracts) . All components except the enzyme were mixed together, the enzyme was added to start the reaction, and the reaction solution was incubated at 30 ° C (gentle shaking) for 90 minutes. Reactions were performed in duplicate, with negative controls where no enzymes were added. The reaction was stopped by the addition of 10% formic acid (final concentration), the mixture was centrifuged at 21,000 rpm, and the supernatant was carefully removed and filtered. Data were corrected for background glutamate levels and for dilution from addition of acid to precipitate proteins, then normalized by the amount of mutant aminotransferase added. When tryptophan was used as a substrate, HEXaspC produced 13.0 mM glutamate per mg aminotransferase per hour. The relative activity, expressed as a percentage of the mutants, is as follows: HEXaspCW130F (156%), HEXaspCT156A (151%), HEXaspCP9T / T156A (63.7%), HEXaspCP9T / R122G (116%) and HEXaspCR122G / T156A (107%) ). When (S, S) -monatin was used as the substrate, HEXaspC produced 7.43 mM glutamate per mg aminotransferase per hour. The relative activity, expressed as a percentage of the mutants, is as follows: HEXaspCW130F (113%), HEXaspCT156A (87.7%), HEXaspCP9T / T156A (67.3%), HEXaspCP9T / R122G (11.2%) and HEXaspCR122G / T156A (114%).

The HEXaspCP9T / R122G mutant exhibited superior activity for the conversion of tryptophan to indole-3-pyruvate, but inferior activity for transamination of (S, S) -monatin. The tryptophan ratio for monatin activity was 18.2 compared to 1.75 for HEXaspC, making it a desirable candidate for the production of (R, R) -monatin with the use of pathways that require an L -aminotransferase as described in Examples 18 and 19. As such, HEXaspCP9T / R122G is an example of an aminotransferase with limited activity on (S, S) -monatin (and MP).

Most mutations improved L-tryptophan activity, but only two mutants increased L-tryptophan activity and (S, S) -monatin (HEXaspCW130F and HEXaspCR122G / T156A). Because 25 mM of the substrate was used in these assays, the enzymes are more likely to be saturated and the activity is a kcat reflection of the enzymes. However, under the conditions under which the tests for the production of (S, S) -monatin (previously) were carried out, it is unlikely that the concentration of S-MP is sufficient to saturate the enzyme, therefore, it is not reflected the increase in kcat. It has been reported that, for similar substrates, that some of the mutations increased kcat but also increased the apparent Km for the amino acid substrate. By using increased concentrations of the substrates, these two mutants are expected to provide a benefit in (S, S) -monatin production rates compared to HEXaspC. The HEXaspCT156A mutation appears to have higher tryptophan transamination rates without having a significant effect on the MP transamination rate under the above conditions for monatin (S, S) production.

By comparing the structures of HEXaspC and one of the Bacillus sp. D-aminotransferase enzymes. (see, eg, S. Sugio, GA Petsko, JM Manning, K. Soda, and D. Ringe, Biochemistry 34 (1995) pp. 9661-9669), the W130F, R122G, T156A and HEX mutations of AspC can be traced for the corresponding residues in the D-aminotransferase structure. Similar broad-specific Daminotransferase mutations are expected to improve overall production of (R, R) -monatin as described in Example 14. For example, the functionality provided by tryptophan 130 in AspC is replaced by Daminotransferases in Bacillus by hydrogen bonding between the side chains of serines 179 to 181 and glutamate 166 (numbering scheme YM-1). To decrease steric hindrance, glutamate can be mutated to an aspartate residue. Some D-aminotransferases have a threonine residue at position 179, which would increase steric hindrance and should be avoided. The B. sphearicus enzyme has an alanine instead of a serine at position 181, which can also reduce steric hindrance.

Additional information from studies of aspartate aminotransferase may also apply to Daminotransferase. While the AspC enzyme has an arginine at the active site that interacts with the side chain of dicarboxylate substrates, D-aminotransferase has a loop from Ser240 to Ser243. The Ser240, Tr242 and Ser243 side chains follow the same direction and form a pocket with the Ser180 hydroxyl group that provides a surface for nonpolar and polar substrates to interact. Ser180 is involved in PLP binding; however, to enhance the activity of a D-aminotransferase with R-MP, residues of Ser240, Tr242, or Ser243 can be modified to accept larger substrates or to favor negatively charged substrates. For example, Tr242 can be mutated to Ser to reduce the length of the side chain. One of the residues can be mutated to lysine or arginine, like Ser243. The Val30-Val36 residues (YM-1 numbering) are located on a beta strand along the active site of D-aminotransferase and are also important for activity. Tir31, Val33, Glu32 and Lis35 are thought to be facing the active site. Tir31, Glu32 and Val33 are invariant in all Bacillus homologs. Ro et al. (FEBS Lett 398 (1996) pp. 141145) mutagenized Val33 to Ala and found a slight increase in catalytic efficiency for alpha-ketoglutarate transamination and significantly higher catalytic efficiency for more bulky substrates (minor steric hindrance). In some homologs Lis35 is replaced with Arg, but steric hindrance is a concern, Lis residue is preferred. Valines 34 and 36 are also preferred over conservative substitutions like isoleucine, again due to less steric hindrance for larger molecules like MP.

Example 17 (not claimed)

Cloning, expression and testing of glutamate and aspartate racemases

This example describes the procedures used to clone and test amino acid racemase enzymes, which can be used to interconvert between L-glutamate and D-glutamate (or L- and D-aspartate or L- and D-alanine). Glutamate, aspartate, or alanine racemases are useful in a biosynthetic pathway to produce (R, R) -monatin when a step in that pathway produces an L-amino acid (such as L-glutamate, L-aspartate, or L-alanine) and another stage in the pathway consumes a D-amino acid (such as D-glutamate, D-aspartate or D-alanine). Figure 4 illustrates a biosynthetic pathway to produce (R, R) -monatin from L-tryptophan using a specific L-tryptophan aminotransferase, a specific R aldolase, a D-aminotransferase, and a glutamate racemase (aspartate or to the girl).

Genes were cloned into vectors pET28 and pET30 to generate the unlabelled proteins and the HIS6-marker / T7-cleavable N-terminal fusion proteins. The resulting proteins were purified using immobilized metal ion affinity chromatography.

Experimental summary

Genes encoding the glutamate racemases (EC 5.1.1.3) from Lactobacillus brevis (Genbank Accession No. D29627, nucleic acid sequence) and Pediococcus pentosaceus (gene MurI) (Genbank Accession No. L22789) were cloned and expressed in E. coli. The extracts were tested to verify the activity in the conversion of L-glutamate to D-glutamate and D-glutamate to L-glutamate. BioCatalytics aspartate racemase enzyme (EC 5.1.1.13) (BioCatalytics, Pasadena, CA) was also tested for interconversion between L- and D-aspartate.

Isolation of Genomic DNA for cloning

L. brevis genomic DNA (ATCC 8287D) was obtained from the American Type Culture Collection. P. pentosaceus (ATCC 25745) was cultured at 37 ° C in lactobacilli MRS broth and 2 ml was used for the isolation of genomic DNA using the Mekalanos J J. method of duplication and amplification of toxin genes in Vibrio cholerae cells. . 1983. 35: 253-63.

Polymerase chain reaction protocol

Primers were designed with 5 'restriction sites and overhangs to clone vectors pET 28 and pET30 (Novagen, Madison, WI).

L. brevis glutamate racemase primers:

Terminal N: 5'-GCGGCGCCATGGAAAATGATCCGATTGGTCTAATG-3 ', (SEQ ID NO: 401), and

Terminal C: 5'-GCGGCGGTCGACGCAATTACAATTGTGTTTGTC-3 '. (SEQ ID NO: 402).

P. pentosaceus glutamate racemase primers:

Terminal N: 5'-GCGGCGCCATGGATGTATGTATAATTTTATTTAG-3 ', (SEQ ID NO: 403), and

Terminal C: 5'-GCGGCGGTCGACAAATTTCATTATTCATTCTAATTT-3 '(SEQ ID NO: 404).

The gene derived from L. brevis was amplified using the following PCR protocol. In a 50 µL reaction 0.150 µg of template, 1.6 µM of each primer, 0.4 mM of each dNTP, 2.8 U of Expand High Fidelity ™ polymerase (Roche, Indianapolis, Ind.) Were used. , 0.5 U of Pfu polymerase (Stratagene, La Jolla, CA) and 1 × Expand ™ buffer with Mg. The thermocycler program used included a warm start at 96ºC for 3 minutes, 8 repetitions of the following stages: 94ºC for 30 seconds, 52ºC for 45 seconds and 72ºC for 2 minutes, followed by 22 repetitions of the following stages: 94ºC for 30 seconds , 60ºC for 45 seconds and 72ºC for 2 minutes. After 22 repetitions the sample was kept at 72 ° C for 7 minutes and then stored at 4 ° C. This PCR protocol produced a product of approximately 830 bp, judging from the comparison with the DNA size markers.

The P. pentosaceus-derived gene was amplified using the following PCR protocol. In a 50 µL reaction 0.15 µg of template, 1.6 µM of each primer, 0.4 mM of each dNTP, 2.8 U of Expand High Fidelity ™ polymerase, 0.5 U of polymerase and 1 × Expand ™ buffer with Mg. The thermal cycler program used included a warm start at 96ºC for 3 minutes, followed by 8 repetitions of the following stages: 94ºC for 30 seconds, 37ºC for 45 seconds and 72ºC for 2 minutes, followed by 8 repetitions of the following stages: 94ºC for 30 seconds, 45ºC for 45 seconds and 72ºC for 2 minutes, followed by 14 repetitions of the following steps: 94ºC for 30 seconds, 55ºC for 45 seconds and 72ºC for 2 minutes. After 14 repetitions the sample was kept at 72 ° C for 7 minutes and then stored at 4 ° C. This PCR protocol produced a product of approximately 840 bp, judging from the comparison with the DNA size markers.

Cloning

The PCR products were purified on 0.8% TAE-agarose gels using the Qiagen gel extraction kit (Qiagen, Valencia, CA). PCR products were quantified using a SmartSpec 3000 ™ spectrophotometer. The products were digested with restriction enzymes NcoI and SalI according to the manufacturer's recommended protocols (New England Biolaboratories, Beverly, MA) and purified on 0.8% TAE-agarose gels using the extraction kit in Qiagen gel (Qiagen, Valencia, CA). Vectors pET28 and pET30 were prepared by digestion with the restriction enzymes NcoI and SalI followed by treatment with serum alkaline phosphatase and purification in 0.8% TAE-agarose gels using the Qiagen gel extraction kit (Qiagen, Valencia, CA).

The digested vectors and inserts were ligated using the DNA Rapid Ligation Kit (Roche, Indianapolis, IN). Approximately 50 ng of the treated insert, 100 ng of the treated vector (3 to 1 insert to vector ratio), 5 U of T4 DNA ligase and 1 × ligation buffer were incubated for 5 minutes at room temperature. The ligation reactions were purified using the Highly Pure PCR Product Purification Kit (Roche, Indianapolis, IN) and used to transform the DH10B electrocompetent cells from

E. coli (Invitrogen, Carlsbad, CA). 10 µl of each ligation reaction was added to 40 µl of DH10B cells and transformed by electroporation using the Bio-Rad II gene driver (Bio-Rad, Hercules, CA) under the following conditions: 2.5 kV, 25 ° F, 200 ohm in a 0.2 cm cuvette. The cells were allowed to recover in 1 mL of SOC medium at room temperature for 1 hour at 37 ° C with stirring at 225 rpm. Cells were plated on LB plates containing kanamycin (50 µg / mL).

Plasmid DNA was purified from the resulting transformants using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with NcoI and SalI. Plasmid sequences that appear to have a correct insertion were verified by dideoxy chain termination DNA sequencing.

Gene expression and analysis

Plasmid DNA, verified by sequence analysis, was subcloned into E. Coli BL21 (DE3) expression hosts (Novagen, Madison, WI). Cultures developed. Plasmids were isolated using the Qiagen miniprep kit (Qiagen, Valencia, CA) and analyzed by restriction enzymes to confirm identity.

Induction in BL21 (DE3) was initially performed with glutamate racemases from L. brevis and P. pentosaceus in vectors pET28 (unlabeled) and pET 30 (labeled with histidine). A temporary study was conducted with cultures developed in 250 mL of kanamycin containing LB (50 mg / L) at an OD600 between 0.5 and 0.6, induced with 100 mM IPTG (isopropyl thiogalactoside) and sampled at 0 and 3 hours after induction. Cells from 600 µL (hour 0) and 275 µL (at 3 hours) were resuspended in sodium dodecyl sulfate buffer containing 2-mercaptoethanol heated at 95 ° C for 10 minutes and cooled. Aliquots of these whole cell protein samples were analyzed by SDS-PAGE using a 4 to 15% gradient gel.

Cell extracts from the 3 hr cultures were also prepared by suspending cell pellets from 5 ml of culture in 0.625 ml of Buggen Reagent from Novagen (Novagen, Madison, WI) containing 0.625 µl of benzase nuclease and 3 µL of group # 3 protease inhibitor mixture (Calbiochem-Novabiochem Corp., San Diego, CA) at room temperature for 20 minutes with light agitation and centrifugation at 16,000 xg to remove cell debris. Supernatants (cell extracts) were loaded onto 4 to 15% gradient gels for analysis of soluble cell proteins.

Cloned 3-hour samples of glutamate racemases from L. brevis and glutamate racemases from P. pentosaceus showed a total and soluble protein that corresponded to the correct size (approximately 31 kDa). The gene product in L. brevis pET30 (histidine labeled) was overexpressed at a higher level and was also more soluble (> 20% soluble protein) than the L. brevis gene product in pET 28 (unlabelled), thus like the gene products of P. pentosaceus in both vectors. The P. pentosaceus gene product showed the same overexpression and solubility in the pET28 and pET30 vectors, which was significantly lower than that observed for the L. brevis gene product in pET30.

Cells from the induced cultures (250 mL) were centrifuged and washed once with 0.85% NaCl. The cell pellets were resuspended in 5 mL / g wet cells of BugBusterTM Reagent (Novagen, Madison, WI) containing 5 µl / ml of set # 3 protease inhibitor mixture (Calbiochem-Novabiochem Corp ., San Diego, CA) and 1 µl / ml of benzase nuclease. The samples were incubated at room temperature for 20 minutes on an orbital shaker. Insoluble cell debris was removed by centrifugation at

16,000 x g for 20 minutes at 4 ° C.

Cell extracts were analyzed to verify glutamate racemase activity using the following protocol. The 400-µL reactions were carried out in 10 mM potassium phosphate (pH 8.0), 0.2 mM DTT and 10 mM L-glutamate or D-glutamate. Reactions were initiated by the addition of 20-100 µL of cell-free extracts and incubated at room temperature. Aliquots of the samples were taken over a period of 1 minute, 5 minutes, 10 minutes, 20 minutes and 1 hour (the zero minute samples served as control reactions). 2M formic acid (25 µL) was added to each aliquot of 40 µL sample to stop the reaction and the precipitated protein was removed by centrifugation. Supernatants were removed and frozen at -80 ° C until analyzed by LC / MS / MS.

The results of the assays of the cell extracts of the induction of pET30 with 100 mM IPTG (3 hours) demonstrate that the enzymes of L. brevis (Genbank access no. BAA06106.1 GI: 468450) and P. pentosaceus (Genbank access No. AAA16761.1 GI: 349029) have significant levels of racemase activity at both glutamate isomers. The P. pentosaceus racemase (20 µL from cell extracts) reached equilibrium between L- and Dglutamate at 10 to 20 minutes from the start with any of the substrates. The L. brevis enzyme (20 µL from cell extracts) also reached equilibrium in approximately 20 minutes.

Partially purified aspartate racemase enzyme (catalog # ASPR-101) purchased from BioCatalytics, Inc. (Pasadena, CA) was analyzed for activity on L-aspartate and D-aspartate using a protocol similar to the one above. The commercial enzyme showed racemase activity in both isomers. Using 0.5-1 mg enzyme, equilibrium was achieved within 20 to 60 minutes.

The three racemases (glutamate racemase from L. brevis, glutamate racemase from P. pentosaceus and aspartate racemase from BioCatalytics) were also analyzed to verify activity in the (S, S) -monatin using the following protocol. The 400-µL reactions were carried out in 10 mM potassium phosphate (pH 8.0), 0.2 mM DTT and 10 mM (S, S) -monatin. Reactions were initiated by the addition of cell-free extracts (L. brevis and P. pentosaceus) or purified enzyme (aspartate racemase from BioCatalytics) and incubated at room temperature. Aliquots of the samples were taken over a period of 1 minute, 5 minutes, 10 minutes, 20 minutes and 1 hour (the zero minute samples also served as samples without enzyme). 2M formic acid (25 µL) was added to each aliquot of 40 µL sample to stop the reaction and the precipitated protein was removed by centrifugation. Supernatants were removed and frozen at −80 ° C until analyzed by LC / MS / MS (Example 1). No decrease in (S, S) -monatin concentration was noted over time nor was there any increase in (S, R) -monatin (initially present as a contaminating by-product <5%, even in the control without enzymes ). Consequently, none of the racemases analyzed showed activity with respect to monatin.

Example 18 (comparative example)

Production of (R, R) -monatin from L-tryptophan using glutamate or aspartate racemases

This example describes the procedures for producing stereoisomerically enriched (R, R) -monatin from L-tryptophan using an L-tryptophan (L-tyrosine or aromatic) aminotransferase, ProA aldolase, glutamate or aspartate racemase and D-amino acid broad specificity aminotransferase. Figure 5 is a diagram illustrating the path. This approach to stereoisomerically enriched (R, R) -monatin production requires an enzyme for step 1 that has reduced activity in monatin production from the monatin precursor (MP). Based on previous results, the gene products tatA of Sinorhizobium meliloti and Rhodobacter sphaeroides described in Example 1 of WO 03/091396 A2 were used.

Materials and procedures

The glutamate racemases from L. brevis and P. pentosaceus were produced in E. coli as described in Example 17. In some cases the His6-labeled version of these enzymes was purified using His-Bind 900 cartridges according to manufacturer's protocols (Novagen, Madison, WI) and desalted to extract imidazole using PD-10 columns (G25 Sephadex, GE Healthcare, Piscataway, NJ). Enzymes were eluted in 25 mM potassium phosphate, pH 8.0. Aspartate racemase (ASPR-101) and D-aminotransferase (AT-103) were purchased from BioCatalytics, Inc. (Pasadena, CA). Tyrosine (aromatic) aminotransferases from S. meliloti and R. sphaeroides were prepared as described in Example 1 of WO 03/091396 A2. The ProA aldolase from Comamonas testosteroni was prepared as described in Example 4 of WO 03/091396 A2. All of the protein assays were performed using a Bio-Rad protein assay according to the manufacturer's protocols (Hercules, CA).

Reduction in amount of (S, S) -monatin produced by using racemases

The reaction mixtures (1 mL volume, performed in duplicate) contained 100 mM of potassium phosphate buffer (pH 8), 2 mM of MgCl2, 0.05 mM of pyridoxal 5'-phosphate (PLP), 200 mM of sodium pyruvate, 5 mM sodium acetoglutarate or oxaloacetate, approximately 280 µg / mL TatA from S. meliloti provided in a cell extract, 1 mg / mL D-aminotransferase (AT-103) from BioCatalytics (BioCatalytics, Pasadena , CA), 100 µL / mL of cellular extract of glutamate racemase or 1 mg / mL of aspartate racemase and approximately 100 µg / mL of ProA aldolase provided as a cell extract. Solid tryptophan was added at a concentration of 10.2 mg / ml. Negative controls did not contain racemase. The mixtures were incubated at 30 ° C (shaking at 250 rpm) for 1 hour, 2 hours or overnight. The samples were centrifuged to extract the precipitate, filtered by syringe and stored at −80 ° C before monatin analysis using the LC / MS / MS procedure described in Example 1. Most of the samples contained> 95 % of (S, S) -monatin, due to the amounts of native L-aminotransferase present in cell extracts. However, the samples containing racemase had a reduced amount of total monatin as a result of racemase enzymes, which made L-glutamate less available for transamination of MP. Without racemase, monatin of 15452355 ppm (predominantly S, S) was produced during the time span. With the racemases present, only monatin (aspartate racemase) of 340-879 ppm (L. brevis enzyme), 444-531 ppm (P. pentosaceus enzyme) and 506-1460 ppm was produced. These data indicate that the racemases are active under the reaction conditions required to produce monatin. To minimize the formation of (S, S) -monatin from cell extract enzymes such as aspartate aminotransferases, additional experiments were performed with purified enzymes and a higher ratio of D-aminotransferase to L-aminotransferase enzymes.

Conversion of L-tryptophan to monatin isomers containing 4-R

The foregoing experiments were repeated using approximately 54 µg of purified L-aminotransferase (either S. meliloti TatA or R. sphaeroides), 1 mg aspartate aminotransferase (BioCatalytics, Pasadena, CA), 1 mg D -aminotransferase, oxaloacetate as the amino acceptor and 75 µg of purified aldolase. Reactions were performed in duplicate with a sampling time of 2 hours and an incubation time overnight. Negative controls were performed with S. meliloti L-aminotransferase but not racemase.

In addition to the quantification of the R, R / S, S and S, R / (R, S) -monatin peak based on reverse phase chromatography, the percentage of each stereoisomer was determined by using the derivatization technique of the FDAA described in Example 1. The results are as follows:

Table 22

L-Aminotransferase

Incubation time Total monatin (ppm) % H.H % R, R % R, S % MR

TatA of S. meliloti

2 hours 17.1 10.2 58.1 0.8 31.0

TatA of S. meliloti

2 hours 15.8 13.3 55.3 1.0 30.4

TatA of S. meliloti

overnight 77.7 25.8 40.0 1.3 32.9

TatA of S. meliloti

overnight 67.9 29.4 37.3 1.5 31.8

TatA of R. sphaeroides

2 hours 241.2 96.3 2.3 0.8 0.6

TatA of R. sphaeroides

2 hours 223.2 95.7 2.7 1.0 0.6

TatA of R. sphaeroides

overnight 600.4 96.6 1.8 0.5 1.1

TatA of R. sphaeroides

overnight 618.5 96.1 2.1 0.5 1.3

no racemase control

2 hours 7.1 92.0 1.4 6.6 0.0

no racemase control

2 hours 5.7 94.0 1.2 4.8 0.0

no racemase control

overnight 44.6 93.5 1.3 4.7 0.5

no racemase control

overnight 37.5 95.4 0.9 3.7 0.0

The presence of racemase clearly increased the total amount of monatin produced when TatA from S. meliloti was used as the enzyme for transamination of L-tryptophan. Monatin levels increased from an average of 6.4 to 16.5 ppm in the 2-hour test and from 41 to 73 ppm in the overnight test. Furthermore, the percentage of R, R formed increased from about 1% to as much as 58% by use of the enzyme racemase. The monatin S, R stereoisomer, another powerful sweetener, was the other major component, increasing from nearly 0 in negative controls to 31%. TatA from R. sphaeroides showed more activity in S-MP than L-transaminase from S. meliloti, which showed the importance of having an enzyme that has a high substrate specificity for L-tryptophan compared to MP when the 4-R isomers of monatin are the desired products. As approximately 10% of the total monatin was 4S in the time interval of two

15 hours, the S. meliloti TatA could be considered to have limited activity in the MP.

The experiments were repeated with the purified S. meliloti TatA (54 µg) and the L. brevis glutamate racemase. When purified glutamate racemase was used, approximately 64 µg per 1 mL reaction was used. Cell extracts containing glutamate racemase were also tested and 1.4 mg of soluble protein were used. A negative control without racemase was used again and all samples were made in duplicate.

20 The results are as follows:

Table 23

Glutamate racemase

Incubation time Total monatin (ppm) % H.H % R, R % R, S % MR

Glutamate racemase

Incubation time Total monatin (ppm) % H.H % R, R % R, S % MR

L. brevis (purified)

2 hours 3.3 49.1 34.2 3.7 13.0

L. brevis (purified)

2 hours 3.6 47.9 35.2 3.5 13.4

L. brevis (purified)

overnight 29.3 58.9 24.7 3.2 13.2

L. brevis (purified)

overnight 40.2 55.1 25.0 4.7 15.3

L. brevis (cell extract)

2 hours 10.5 45.8 35.9 1.1 17.2

L. brevis (cell extract)

2 hours 10.5 47.4 33.9 1.1 17.6

L. brevis (cell extract)

overnight 79.4 70.3 17.9 1.3 10.5

L. brevis (cell extract)

overnight 80.1 69.1 19.1 1.1 10.7

none

2 hours 2.7 84.1 7.1 6.3 2.4

none

2 hours 3.2 84.9 6.0 6.8 2.2

none

overnight 36.5 92.3 2.3 4.2 1.2

none

overnight 30.5 92.7 2.0 4.1 1.3

Again, it is clear that the addition of racemase increases the total monatin produced from L-tryptophan, as well as increases the relative amounts of 4R-containing monatin isomers compared to (S, S) monatin. The use of aldolase, racemase and purified L-aminotransferase greatly increases the capacity of

Control of desired stereoisomer formation. The ratio of L to D aminotransferase is also a way to manipulate the stereochemistry of the final product.

Comparing the results shown in Tables 18 and 19 in Example 13 with the results with reaction conditions similar to the above conditions, one can see that approximately 7 to 29 ppm monatin was formed from indole-3-pyruvate and the percentages of (R, R) -monatin formed were from about 10 to 51 to 90%. The use of aspartate racemase increased the total amount of monatin produced from 16 to 78 ppm monatin with a% R, R from about 40 to 58%. In addition, a more stable and less expensive raw material, L-tryptophan, was used. In Example 14, approximately 73 ppm monatin was produced from D-tryptophan in an R, R: S, R ratio of approximately 1.7: 1. The total amount of 4R isomers was> 80% of the total monatin. Because R, R-monatin and S, R-monatin are powerful sweeteners (1000

15 times sweeter than sucrose) the ability to enrich these isomers without the need for expensive Daminoacid substrates is critical.

As described in Examples 13 and 14, D-alanine can serve as an amino donor for transamination of MP to monatin. Many L-aminotransferases have the ability to use pyruvate as an amino acceptor to some extent and to produce L-alanine. Because the reactions mentioned above use high concentrations of pyruvate, a certain amount of pyruvate is likely to convert to L-alanine. For example, during transamination of L-tryptophan, the enzyme HexAsp described in Example 16 was found to convert 10 to 18% of pyruvate (initial concentrations 50 to 200 mM) to L-alanine in 2 hours if the Alpha ketoglutarate is absent. The enzyme showed a 10-fold preference for alpha ketoglutarate when both amino acceptors were present in higher concentrations (> 50mM). AspC (described in WO 25 03/091396 A2) also produced a certain amount of L-alanine from pyruvate. Therefore, it is expected that the addition of alpha ketoglutarate or oxaloacetate can be omitted in the above reactions and use alanine racemase (EC 5.1.1.1) instead of glutamate or aspartate racemase. Alanine racemase enzymes were first identified in

Brucella abortus and Streptococcus faecalis (Marr, A. G. and Wilson, P. W. Arch. Biochem. Biophys., 49 (1954) 424-433 and Wood, W. A. and Gunsalus, I. C. J. Biol. Chem., 190 (1951) 403-416). The dadB gene in Salmonella typhimurium was identified as the source of alanine racemase activity and several hundred homologs can be found in genomic databases. Other known sources of alanine racemase activity are Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Schizosaccaroyces pombe and Bacillus cereus. The basidiomycete fungus, Lentinus edodes, also contains a highly active alanine racemase. A thermostable Bacillus stearothermophilus homolog is available for purchase from Sigma-Aldrich (Catalog # A8936) (Sigma-Aldrich, St. Louis, MO) and has been immobilized for commercial applications (Inagaki, K., Biochemistry, 25: 3268. 1986). Alanine racemase is converted to a glutamate or aspartate racemase by random procedures such as mutagenic PCR, passage through mutagenic strains, or other procedures known to those skilled in the art. A more focused evolution of the alanine racemase focuses on active site residues, including Lis129, Met134, and residues including between Gli283 and Trp288 (Bacillus stearothermophilus numbering).

Example 19 (comparative example)

D-phenylglycine aminotransferase (D-4-hydroxyphenylglycine aminotransferase)

As shown in Figure 3, D-phenylglycine aminotransferase is useful in a biosynthetic pathway for monatin production. For example, D-phenylglycine aminotransferase produces (R, R) -monatin from R-MP with Lglutamate as the amino donor.

PCR synthesis of P. stutzeri 4 D-hydroxyphenylglycine aminotransferase from oligonucleotide primers

This example describes the procedures that were used to synthesize 4 D-hydroxyphenylglycine aminotransferase, a stereoinverting enzyme that can be used to convert the monatin precursor R to (R, R) -monatin by using L-glutamate as the amino donor.

Primer design

The published sequence (Genbank Access No. AY319935, nucleic acid sequence; Genbank Access No. AAQ8290, protein sequence) for Pseudomonas stutzeri 4-D-hydroxyphenylglycine aminotransferase (4-D-HPG AT) was used as a template for primer design. PCR. Alternatively, the 4-D-hydroxyphenylglycine aminotransferase from Pseudomonas putida, (CAD42450 (protein), AX467211 (nucleotide)) is used as a sequence template. A total of 34 forward primers and 35 reverse primers were designed; forward and reverse primers were 40 primers that shared 20 pairs of overlapping base. In addition, 2 external primers with 5 'restriction sites and overhangs were designed to clone vectors pET 28 and pET30 (Novagen, Madison, WI).

P. stutzeri 4 D-HPG AT external primers: Terminal N (with NdeI site):

5'-GGCCGGCATATGTCGATCCTTAACGACTACAAACGT-3 '(SEQ ID NO: 405) and terminal C (with XhoI site):

5'-GGAAGGCTCGAGTCATGATTGGTTTCCAGACAAATT-3 '(SEQ ID NO: 406).

Polymerase chain reaction protocol

The P. stutzeri gene sequence was amplified using the following protocols. The 100 µL primary PCR reaction included 0.05 µM of each of the 69 internal primers, 0.4 mM of each dNTP, 10 U of rTth Polymerase XL (Roche, Indianapolis, IN), 0.625 U of Pfu polymerase (Stratagene, La Jolla, CA), 1 × XL buffer and 1 mM Mg (OAc) 2. The thermal cycler program used included a warm start at 94ºC for 3 minutes, 15 repetitions of the following stages: 94ºC for 30 seconds, 42ºC for 30 seconds and 68ºC for 15 seconds, followed by 10 repetitions of the following stages: 94ºC for 30 seconds , 52ºC for 30 seconds and 68ºC for 30 seconds, followed by 10 repetitions of the following steps: 94ºC for 30 seconds, 60ºC for 30 seconds and 68ºC for 1 minute and 15 seconds. After the final 10 cycles the sample was kept at 68 ° C for 7 minutes and then stored at 4 ° C. This PCR protocol produced a smear of product at approximately 0.5 kb on a 0.8% TAE-agarose gel.

The secondary PCR reaction was configured using the primary PCR reaction as a template. The 100 µL secondary PCR reaction included 2.5 µL of the primary PCR reaction, 0.5 µM of each of the 2 outer primers (with NdeI and XhoI restriction sites), 0.4 mM of each dNTP, 10 U of rTth Polymerase XL, 0.625 U of Pfu polymerase, 1 × XL buffer and 1 mM Mg (OAc) 2. The thermocycler program used included a warm start at 94ºC for 3 minutes, 10 repetitions of the following stages: 94ºC for 30 seconds, 52ºC for 30 seconds and 68ºC for 1 minute and 30 seconds, followed by 15 repetitions of the following stages: 94ºC for 30 seconds, 60ºC for 30 seconds and 68ºC for 1 minute and 30 seconds. After repetitions the sample was kept at 68 ° C for 7 minutes and then stored at 4 ° C. This PCR protocol produced a particular product band at approximately 1.4 kb on a 0.8% TAEagarose gel.

PCR products were purified on 0.8% TAE-agarose gels using the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA). The product was cloned into TOPO and transformed into TOP 10 cells according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from the resulting transformants using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with NdeI and XhoI. Plasmid sequences that appear to have correct insertion were verified by dideoxy chain termination DNA sequencing with the forward M13 and reverse M13 universal primers. Of the 10 sequenced clones, all had at least one mutation of the desired sequence. The best clone had a single base pair mutation that resulted in an amino acid change. The sequence of this clone was corrected using the QuickChange mutagenesis protocol according to the manufacturer's recommendations (Stratagene, La Jolla, CA).

The corrected TOPO clone was digested with restriction enzymes NdeI and XhoI according to the manufacturer's recommended protocols (Biolaboratories of New England, Beverly, MA) and purified on 0.8% TAEagarose gels using the extraction kit. gel from Qiagen (Qiagen, Valencia, CA). Vectors pET28 and pET30 were prepared by digestion with restriction enzymes NdeI and XhoI followed by treatment with serum alkaline phosphatase and gel purification of 0.8% TAE-agarose using the Qiagen gel extraction kit (Qiagen , Valencia, CA).

The digested vectors and inserts were ligated using the NEB Rapid Ligation Kit (Beverly, MA). Approximately 50 ng of the treated insert, 100 ng of the treated vector (3 to 1 ratio of insert to vector), 5 U of T4 DNA ligase and 1 × ligation buffer were incubated for 5 minutes at room temperature. The ligation mixture was transformed into TOP10F 'chemically competent cells (Invitrogen, Carlsbad, CA). The cells were allowed to recover in 0.25 mL of SOC medium at room temperature for 1 hour at 37 ° C with stirring at 225 rpm. Cells were plated on LB plates containing kanamycin (50 µg / mL). Plasmid DNA was purified from the resulting transformants using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with NdeI and XhoI.

Gene expression and analysis

Plasmid DNA was transformed into E. Coli BL21 (DE3) expression hosts (Novagen, Madison, WI). Cultures were grown and plasmids were isolated using the Qiagen miniprep kit (Qiagen, Valencia, CA) and analyzed by restriction enzymes to confirm identity.

BL21 (DE3) induction was initially performed with P. stutzeri 4 D-HPG in vectors pET 28 (histidine labeled) and pET 30 (unlabeled). A temporary study was performed with cultures developed in 250 mL of kanamycin containing LB (50 mg / L) at an OD600 of between 0.5 and 0.6, induced with 100 mM IPTG (isopropyl thiogalactoside) and sampled at 0 and 3 hours after induction. A suitable volume of cells from hour 0 and hour 3 was resuspended in 40 µL of sodium dodecyl sulfate buffer containing 2-mercaptoethanol and heated at 95 ° C for 10 minutes and cooled. Aliquots of these whole cell protein samples were analyzed by SDS-PAGE using a 4 to 15% gradient gel.

Cell extracts from the 3 hr cultures were also prepared by suspending cell pellets from 5 ml of culture in 0.625 ml of Buggen Reagent from Novagen (Novagen, Madison, WI) containing 0.625 µl of benzase nuclease and 3 µL of Joint Protease Inhibitor Mix # 3 (Calbiochem-Novabiochem Corp., San Diego, CA) at room temperature for 20 minutes with light agitation and centrifugation at 16,000 xg to remove cell debris. Supernatants (cell extracts) were loaded onto 4 to 15% gradient gels for analysis of soluble cell proteins. When required, the protein was purified using His-Bind 900 cartridges according to the manufacturer's protocols (Novagen, Madison, WI) and desalted to extract the imidazole using PD-10 columns (G25 Sephadex, GE Healthcare, Piscataway, NJ).

Organisms with D-phenylglycine aminotransferase (DPGAT)

Organisms of the genus Pseudomonas and similar genera, with a stereoinverting D-phenylglycine aminotransferase (also called D-4-hydroxyphenylglycine aminotransferase) are isolated as follows. The soil samples are incubated in petri dishes with the following medium: (per liter) 15 g of Agar, 3.4 g of KH2PO4, 3.55 g of Na2HPO4, 0.2 g of MgSO4.7H2O, 8 mg of CaCl2 .2H2O, 10 mg of yeast extract, 1 ml of 1000 × trace element solution, 1 g of D-phenylglycine (D-4-hydroxyphenylglycine).

Isolates were tested by PCR to detect the presence of a stereoinverting aminotransferase (primers designed from known D-phenylglycine aminotransferases) or further enriched to detect the presence of a stereoinverting aminotransferase as follows: the isolates from the plates were Cultured in liquid medium as above minus agar at 30 ° C with shaking at an OD600 of about 1.0. Cells were harvested by centrifugation and washed twice with 0.85% NaCl. A 10 mg (wet weight) sample was suspended in 1 ml of potassium phosphate buffer (pH 7.0) and 5 mM of D-phenylglycine (or D-4-hydroxyphenylglycine). Neutralized 15 mM acetic acid (aminooxy) was added to duplicate the samples prepared as described above. The consumption of D-phenylglycine (or D-4-hydroxyglycine) was measured by HPLC. Isolates capable of degrading D-phenylglycine (or D-4-hydroxyphenylglycine), but which did so to a lesser extent in the presence of acetic acid (aminooxy), were selected for further analysis. Isolates were tested by PCR to verify the presence of stereoinverting aminotransferase (primers designated from known D-phenylglycine aminotransferases).

The presence of stereoinverting aminotransferase was confirmed by developing a culture in a liquid medium as described above, harvesting the cells and preparing a crude cell-free extract (CFE) and the test to detect the enzymatic activity of D -phenylglycine aminotransferase (or D-4-hydroxyphenylglycine aminotransferase). CFE was added to the reaction mixture with the following final concentrations: 0.1M CAPS (pH 9.5), 60mM L-glutamate (sodium salt), 5mM benzoylformate (or 4-hydroxybenzoate) and 50 ! M from PLP. The reverse reaction was measured by adding CFE to the reaction mixture at the following concentrations: 50mM potassium phosphate (pH 7.0), 60mM D-phenylglycine (or D-4-hydroxyphenylglycine), 5mM of a-ketoglutarate, 50 µM of PLP. Assays were incubated at 35 ° C and aliquots were taken at time intervals and boiled for 2 minutes. The product will be quantified by the HLPC procedure of Gil-Av, E., Tishbee, A., Hare, P. E., Resolution of underivatized amino acids by reversed phase chromatography.

J. Am. Chem. Soc., 102: 5115-5117 (1980) or by the procedures described in Example 1 (measurement of glutamate formation).

As an alternative to PCR-based procedures, stereoinverting D-phenylglycine aminotransferase is purified from the isolated bacterium by standard protein purification techniques including ammonium sulfate fractionation and standard column chromatography. Once the protein has been purified to a reasonable degree, peptide microsequencing or conventional Edman-type amino acid sequencing techniques are used (see golgi.harvard.edu/microchem/ for descriptions of the protocols and equipment used for this type of work. ). Degenerate primers are designed based on the sequence available from the closest known relative of the protein source. Degenerate PCR and genomic walk are then performed according to established protocols to isolate the sequence encoding the stereoinverting D-phenylglycine aminotransferase.

DPGAT monatin production

D-hydroxyphenylglycine aminotransferases, as described in (1) and (2) above, are used in crude or purified protein-free extracts as described in (1) above. The S. aromiloti and R. sphaeroides tyrosine (aromatic) aminotransferases were prepared as described in Example 1 of WO 03/091396 A2. The ProA aldolase from Comamonas testosteroni was prepared as described in Example 4 of WO 03/091396 A2. All of the protein assays were performed using a Bio-Rad protein assay according to the manufacturer's protocols (Hercules, CA).

The reaction mixtures (1 mL volume, performed in duplicate) contained 100 mM of potassium phosphate buffer (pH 8), 2 mM of MgCl2, 0.05 mM of pyridoxal 5'-phosphate (PLP), 200 mM of sodium pyruvate, 5 mM sodium acetoglutarate, approximately 280 µg / mL of S. meliloti TatA provided in a cell extract, 100 µL / mL of D-hydroxyphenylglycine aminotransferase cell extract or 1 mg / mL of D- Purified hydroxyphenylglycine aminotransferase and approximately 100 µg / mL ProA aldolase provided as a cell extract. Solid tryptophan was added at a concentration of 10.2 mg / ml. Negative controls were configured without Dhydroxyphenylglycine aminotransferase. The samples were incubated at 30 ° C with gentle shaking for approximately 1 hour or overnight. The samples were centrifuged to extract the precipitate, filtered by syringe and stored at -80 ° C before monatin analysis using the LC / MS / MS procedure described in Example 1.

D-hydroxyphenylglycine aminotransferases with increased activity for monatin production are prepared by mutagenesis techniques known to those skilled in the art, including: mutagenic PCR, passage through mutagenic strains, or site-directed mutagenesis. Enhanced D-hydroxyphenylglycine aminotransferases are selected for growth in minimal medium with R, R-monatin as the nitrogen source. Initially selection was based on growth but as the improved aminotransferases were selected the analysis was based on growth rate. That is, cells were cultured with mutated versions of the gene, and the gene was expressed in minimal medium with R, R-monatin as the nitrogen source. Cell growth rates with the mutated versions of the gene were compared to the unmutated version. Cells with a faster growth rate were selected and aminotransferase was further analyzed. D-hydroxyphenylglycine aminotransferase was mutagenized by available techniques such as error-prone PCR and passage through mutagenic strains or by procedures for which a license such as DNA restructuring and other directed evolution technologies has been obtained.

DPGAT test

Cells were cultured with recombinant D-hydroxyphenylglycine aminotransferase, the protein was expressed, and cells were lysed as described in Example 17 or by standard protocols. The protein was expressed in its

native receptor by using the procedures described (Wiyakrutta, W., Meevootisom, V. A stereoinverting D-phenylglycine aminotransferase from Pseudomonas stutzeri ST-201: purification, characterization, and application for D-phenylglycine synthesis. J. Biotechnol., 55 : 193-203 (1997)).

Cell-free extract was added to the reaction mixture with the following final concentrations: 100 mM potassium phosphate (pH 7.0 to 8.5), 60 mM D-phenylglycine (or D-4-hydroxyphenylglycine), 5 a-ketoglutarate mM, 50 µM PLP. The tests were incubated at room temperature, aliquoted at time intervals and stopped by adding an equal volume of formic acid. The product (L-glutamate) was quantified by the procedures described in Example 1.

Example 20 (comparative example)

Discovery of a D-methionine aminotransferase gene

Background

D-methionine pyruvate aminotransferase (EC 2.6.1.41) is considered to be another, albeit rare, example of a stereoinverting transaminase. This enzyme catalyzes the reversible conversion of D-methionine and pyruvate to Lalanine and 4-methylthio-2-oxobutanoate. Oxaloacetate, phenylpyruvate, 2-oxobutyrate, 2-oxovalerate, 2-oxoheptanoate, glyoxylate, and oxoglutarate can also serve as amino acceptors.

Transamination of D or L methionine is thought to be part of a pathway for the production of ethylene in higher plants (cauliflower, tomato, apple, pea, banana, peanut) as well as microorganisms in the soil (Escherichia coli, Pseudomonas pisi, Pseudomonas aeruginosa, Bacillus mycoides, Acinetobacter calcoaceticus, Aeromonas hydrophila B12E, Rhizobium trifolii N2P7, Penicillium digitatum, Saccharomyces cerevisiae, Corynebacterium D7F).

D.C. Billington, B. T. Golding, and S. B. Primrose Biochem J. (1979) 182, 827-836. In bacteria, L-methionine is typically used as the substrate in ethylene production studies and broadly specific enzymes such as TyrB or AspC from E. coli are thought to be responsible for transamination. However, S. B. Primrose J. Gen. Microbiol. (1976), 95 (1), 159-65 and S. B. Primrose J. Gen. Microbiol. (1977), 98, 519-528 showed that the E. coli strain SPA O (University of Warwick culture collection) produced almost as much ethylene from D-methionine as from L-methionine in batch cultures . Because no broadly specific Daminotransferase was identified in E. coli, one possible explanation may be that E. coli D-amino acid dehydrogenase (encoded by the dadA gene) converts D-methionine to 4-methylthio-2 -oxobutanoate. It is also possible that a methionine racemase exists in E. coli; however, no such enzyme has been described in the literature.

Contrary to E. coli, in cauliflower heads (mitochondrial extract preparations) and in peanut sprouts, ethylene production was higher when D-methionine and pyruvate were provided to the enzyme extract compared to L-methionine and pyruvate (LW Mapson, JF March, and DA Wardale Biochem J (1969) 115, 653-661; JI Durham, PW Morgan, JM Prescott and d CM Lyman Phytochemistry (1973) 12, 21232126). Therefore, the possibility of a combination of methionine racemase and an L-aminotransferase is not supported by the data. Dehydrogenase activity was ruled out by dialysis of the cauliflower cell extracts, no NAD was found in the test mixtures. Oxidase activity was ruled out since no oxygen consumption was detected and there was no FAD requirement. D-methionine aminotransferase from peanut tissues was purified, shown to be dependent on PLP and shown to be independent of L-methionine aminotransferase activity. There is a possibility that these D-methionine-pyruvate aminotransferases actually produce D-alanine as a by-product (similar to the Bacillus enzymes described in Examples 13 and 14) and that cells contain alanine racemase to recycle D-alanine back to L-alanine (or an amino analog donor). In either case, the discovery of broadly specific D-aminotransferase in taller plants is advantageous for the development of processes that produce R, R monatin or (S, R) -monatin.

Experimental summary

D-methionine aminotransferase is partially purified from cauliflower heads and germ peanut embryos using standard chromatography protocols and the Pharmacia AKTA Explorer system. Protein sequences of homologous proteins are determined by LC / MS / MS fingerprint techniques and database search by the Harvard Microchemistry Center. The coding regions of the plant genes are cloned from the cDNA library using standard PCR protocols or by gene synthesis as described in Example 19.

Alternatively, cDNA expression libraries are constructed (Stratagene, La Jolla, CA) from cauliflower tissue or groundnut seeds grown in the presence of D-methionine (and with ethylene production). Libraries are transformed into E. coli methionine auxotrophs from the E. coli genetic lineage center (Yale) as strains RC519 or AB1931. The plasmids from the strains capable of growing in minimal medium containing D-methionine contain the coding region of interest (see Example 15, an analogous analysis technique).

Once the coding regions of interest are obtained and expressed in a standard laboratory strain of

E. coli, the resulting gene products can be used in assays to produce (R, R) -monatin as described in Example 19 in place of D-hydroxyphenylglycine aminotransferase, with the exception of the pH being 7.5 (the Optimal pH for aminotransferase). If D-methionine aminotransferase has a strict requirement for D-amino acid donor substrates, the enzyme can be used to prepare (R, R) -monatin as described in Examples 13 and 14. Genes can be mutagenized and analyzed to detect increased activity as described in Example 19.

Procedures

Cauliflower insulation

400 grams of freshly collected cauliflower heads were extracted with 400 mL of a sucrose / buffer solution at 4 ° C (0.4M sucrose and 0.1M sodium phosphate buffer, pH 7.4) by alternating soaking and mixing with the use of a mixer. The cell debris was filtered off with cheesecloth and the resulting solution was centrifuged at 40,000 × g for 30 min at 4 ° C. The solid material (containing mitochondrial organelles) was resuspended in 20 mL of 10mM sodium phosphate buffer, pH 7.4, and the enzymes were extracted with 200 mL of cold acetone (−30 ° C). The suspension was recentrifuged and the precipitate was dried using Savant Speed Vac. The solid material was dissolved in 10 mM sodium phosphate buffer, pH 7.4, and the residual acetone was extracted using a PD-10 column (GE Healthcare, Piscataway, NJ).

Aminotransferase activity was analyzed by incubating the enzyme preparation with 5mM Dmethionine, 1mM pyruvate, 0.05mM PLP and 2mM EDTA in 0.1M sodium phosphate buffer, pH 7.4. The tests were carried out at 25 ° C for 16 hours. 4-Methylthio-2-oxobutanoate was measured through the formation of the 2,4-dinitrophenylhydrazone derivative, using LC / MS (m / z 328) and similar methodologies described in Example 1. A 0.4% (w / v) solution of 2,4-dinitrophenylhydrazine in 2M sulfuric acid and half the volume was added to the test mixture after incubation. The mixture was mixed with gentle stirring at 30 ° C for 30 minutes and the precipitate was collected by centrifugation and analyzed by LC / MS. Protein fractions separated by standard chromatographic techniques were analyzed to verify activity in a similar way, but the co-product alanine was measured by the OPA post-column derivatization technique described in Example 1.

Peanut Isolation (Arachia hypogea L. cv. Starr)

The enzyme D-methionine aminotransferase from the germ peanut embryo homogenate (minus cotyledons) is purified according to the procedure of J. I. Durham, P. W. Morgan, J. M. Prescott and C. M. Lyman Phytochemistry (1973) 12, 2123-2126. Reducing agents are used during the preparation of crude extracts to stabilize the enzymes and the cell debris is removed by centrifugation at 33,000 × g. The 35 to 50% ammonium sulfate fraction is further purified by incubation at low temperature and by removal of proteins in the precipitate. The supernatant is further fractionated using acetone. The active groups below are further purified by gel filtration chromatography (Sephadex 200, GE Healthcare, Piscataway, NJ).

As protein fractions are enriched with protein transaminase, 2D gel electrophoresis is used to separate the enzyme of interest for microsequencing. After elucidation of the homologs, the coding regions in the plant sequences deposited in NCBI, the protein D-aminotransferase is produced recombinantly in Escherichia coli by using standard molecular biology techniques. It is expected that the cell extracts of cauliflower heads or peanut seeds or recombinantly produced homologous enzymes can be used in the production of (R, R) -monatin as described in Example 19 (if it is a stereoinverting transaminase ) or in Examples 13 and 14 (if it is a broad specificity D-aminotransferase).

Example 21 (comparative example)

L-alanine aminotransferase / alanine racemase / D-alanine aminotransferase

Figures 4, 6 and 8 illustrate the biosynthetic pathways for producing stereoisomerically enriched (R, R) -monatin from L-tryptophan using an L-amino acid aminotransferase (such as L-alanine-aminotransferase and / or L-tryptophan -aminotransferase), an R-specific aldolase, an alanine racemase and a D-alanine aminotransferase.

Example 16 describes a tryptophan-specific aminotransferase. Alternatively, the (aromatic) tyrosine aminotransferases of S. meliloti and R. sphaeroides were prepared as described in Example 1 of WO 03/091396 A2. The ProA aldolase from Comamonas testosteroni was prepared as described in Example 4 of WO 03/091396 A2. All of the protein assays were performed using a Bio-Rad protein assay according to the manufacturer's protocols (Bio-Rad, Hercules, CA). Alanine racemase was purchased from Sigma-Aldrich (St. Louis, MO) (catalog number A8936). D-Alanine Aminotransferase was purchased from BioCatalytics (Catalog Number AT-103) (BioCatalytics, Pasadena, CA).

L-alanine aminotransferases are widely distributed in eukaryotes, bacteria, and archaea. The following organisms (based on sequence homology) were identified as containing an L-alanine aminotransferase (EC 2.6.1.2): Arabidopsis thaliana, Ashbya gossypii, Azotobacter vinelandii, Bifidobacterium longum, Caenorhabditis elegans, Candida albicans, Candida glabrata, Chlamydom reinhardtii, Cryptococcus neoformans, Debaryomyces hansenii, Homo sapiens, Hordeum vulgare, Kluyveromyces lactis, Magnaporthe grisea, Medicago truncatula, Mus musculus, Neurospora crassa, Pyryasrocous, Pyus taedacoccus, Pus taedarocous Rattus norvegicus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Takifugu rubripes, Trypanosoma cruzi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yarrowia lipolytica and Zea mays. Furthermore, many aminotransferases have a low level of alanine aminotransferase activity and given the high levels of L-glutamate and pyruvate they can be converted to L-alanine and a-ketoglutarate. The enzyme with low activity is improved with standard mutagenesis techniques such as error-prone PCR and passage through mutagenic strains or through directed evolution techniques. The gene for an L-alanine aminotransferase is cloned by using publicly available designer primers and by using standard techniques to amplify, clone, express and purify the gene / enzyme.

The reaction mixtures (1 mL volume, performed in duplicate) contained 100 mM of potassium phosphate buffer (pH 8), 2 mM of MgCl2, 0.05 mM of pyridoxal 5'-phosphate (PLP), 200 mM of sodium pyruvate, 5 mM sodium acetoglutarate, approximately 280 µg / mL of S. meliloti TatA provided in a cell extract (or other specific L-tryptophan aminotransferase), 100 µL / mL of alanine racemase cell extract or 1 mg / mL of purified alanine racemase, about 280 µg / mL of D-alanine aminotransferase provided in a cell extract and about 100 µg / mL of ProA aldolase provided as a cell extract. Solid tryptophan was added at a concentration of 10.2 mg / ml. Negative controls were configured without alanine racemase. The samples were incubated at 30 ° C with gentle shaking for approximately 1 hour or overnight. The samples were centrifuged to extract the precipitate, filtered by syringe and stored at −80 ° C before monatin analysis using the LC / MS / MS procedure described in Example 1. In these reaction mixtures, if the L-tryptophan aminotransferase can use α-ketoglutarate but not pyruvate, as the amino acceptor, an L-alanine aminotransferase can be added that converts L-glutamate and pyruvate to L-alanine and acetoglutarate, as shown in figure 6.

Example 22

Subcloning of genes encoding aldolases of SEQ ID NO: 276, 244, 218, 228, 162, 50, 74, 44 and 116

Genes encoding aldolases having the amino acid sequences of SEQ ID NO: 276, 244, 218, 228, 162, 50, 74, 44, and 116 were subcloned into the expression vector pET28b (Novagen, Madison, WI) with N-terminal His markers to allow purification. SEQ ID NO: 275 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 276. SEQ ID NO: 243 is the gene sequence encoding aldolase that has the amino acid sequence of SEQ ID NO: 244. SEQ ID NO: 217 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 218. SEQ ID NO: 227 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 228. SEQ ID NO: 161 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 162. SEQ ID NO: 49 is the gene sequence encoding aldolase that has the amino acid sequence of SEQ ID NO: 50. SEQ ID NO: 73 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 74. SEQ ID NO: 43 is the gene sequence encoding aldolase that has the amino acid sequence of SEQ ID NO: 44. SEQ ID NO: 115 is the sequence of the gene encoding aldolase that has the amino acid sequence of SEQ ID NO: 116.

The primers used for cloning are shown in Table 24.

Table 24

Aldolase DNA SEQ ID NO .:

primer 5 ’ primer 3 ’

275

5'-ATAAGACATATGCCTATCGTTGTTACGAAG3 '(SEQ ID NO: 339) 5'-ATAAGAGGATCCTTATTCCTCGGGCAGCCGCT C-3 '(SEQ ID NO: 340)

243

5'-ATAAGACATATGAACAGACCTGTGGTTGTC -3 '(SEQ ID NO: 341) 5'-ATAAGAGGATCCTTACAGGTACTTGAGACCGA G-3 '(SEQ ID NO: 342)

Aldolase DNA SEQ ID NO .:

primer 5 ’ primer 3 ’

217

5'-ATAAGACATATGAGCGTGGTCATCCGAAAC -3 '(SEQ ID NO: 343) 5'-ATAAGAGGATCCTTACTTCGCTTTGTTATAGG C-3 '(SEQ ID NO: 344)

227

5'-ATAAGACATATGAACAAG (SEQ ID NO: 345) 5'-ATAAGAGGATCCTTACAA (SEQ ID NO: 346)

161

5'-ATAAGACATATGAGCGTGGTCGTCACCGG3 '(SEQ ID NO: 347) 5'-ATAAGAGGATCCTTAGCCGTTTTTCCCGTCGG TG-3 '(SEQ ID NO: 348)

49

5'-AGAAGACATATGATGAGCATCGTCGTCCA GAAC-3 '(SEQ ID NO: 349) 5'-AGAAGAGGATCCTCAGACATATTTCAGGCCCT TG-3 '(SEQ ID NO: 350)

73

5'-AGAAGACATATGATGAGCGTGGTCATCAC C-3 '(SEQ ID NO: 351) 5'-ACAACAGGATCCCTATTTCTTCTCCGGCGTTT C-3 '(SEQ ID NO: 352)

43

5'-ATAATACATATGAGCGTC (SEQ ID NO: 353) 5'-ATAATAGGATCCTTAGAC (SEQ ID NO: 354)

115

5'-AGAAGACATATGATGTCG (SEQ ID NO: 355) 5'-AGAAGAGGATCCTCAGAT (SEQ ID NO: 356)

Genes encoding the above aldolases were amplified by PCR and digested with suitable enzymes (Nde I and BamH I) and gel purified (QiAquick® Gel Extraction Kit (Qiagen, Valencia, CA)). The digests were individually ligated to pET28 which had been digested with Nde I and BamH I and gel purified. The

Ligation was transformed into TOP10 cells (Invitrogen, Carlsbad, CA). DNA miniprep from individual colonies was analyzed for the presence of insertions by size analysis using agarose gel electrophoresis. Isolates with an insert were subjected to DNA sequence analysis (Agencourt, Beverly, MA).

Aldolase purification

Confirmed aldolase clones were transformed into BL21 (DE3) or BL21 (DE3) pLysS. Induction was carried out overnight in Terrific broth at 30 ° C. Cultures grown overnight with the appropriate antibiotic were diluted in fresh medium (typically 1: 100) and grown at an OD600 of approximately 0.6 with aeration at 37 ° C. Cultures were then induced with 1mM IPTG and changed at 30 ° C (with aeration) and incubation continued overnight. Cells were harvested by centrifugation. The cell sediment is

15 typically underwent a freeze-thaw cycle to aid cell lysis. The cell pellet was lysed in BugBuster and Benzonase Nuclease (Novagen, Madison, WI) (according to the manufacturer's protocol). Cell debris was removed by centrifugation. The crude protein extract was applied to a 10 mg HIS-Bind column (Novagen, Madison, WI) that had been prepared according to the manufacturer's protocol. The column was washed and the protein was eluted according to the manufacturer's protocol. The protein

Purified 20 was desalted with PD-10 columns (GE Healthcare, Piscataway, NJ) and eluted in 50mM potassium phosphate buffer, pH 7.5, containing 4mM MgCl2, 200mM NaCl. The protein was concentrated with Amicon centrifuge concentrators (5000 MW cut-off) (Millipore, Billerica, MA). After concentration it was noted that the aldolases of SEQ ID NO: 44, SEQ ID NO: 28 and SEQ ID NO: 276 showed a certain level of precipitation, although the activity was still quite high. Proteins were stored at −80ºC until

25 trial.

Protein assays were performed using the Pierce BCA Kit (Pierce, Rockford, IL) and the Bovine Serum Albumin Microtiter Plate ("BSA") protocol as the standard protein. The Experion Pro260 electrophoresis system (Bio-Rad, Hercules, CA) or SDS-PAGE was used to estimate the percentage of aldolase in the purified sample and to evaluate the expression levels in the soluble cell extract and in the total protein.

30 Test for purified aldolases

The purified aldolases were tested to verify their ability to produce (R, R) -monatin from D-tryptophan and compared to aldolases of SEQ ID NO: 28 and SEQ ID NO: 54 prepared in the same way. The tests were performed in microcentrifuge tubes (in duplicate) with purified protein, using the same concentration of enzyme per test (50 µg / mL) with the exception of SEQ ID NO: 244, for which 5 used 25 µg / mL. SEQ ID NO: 243 was not well expressed and provided lower amounts of purified protein. 2 mg / mL AT-103 from BioCatalytics (BioCatalytics, Pasadena, CA) was used as the D-aminotransferase. The following was added to each 1 mL of reaction mixture: aldolase, 4 mM MgCl2, 50 mM D-tryptophan, Daminotransferase, 200 mM sodium pyruvate, 100 mM potassium phosphate buffer, pH 7.5 and 0 0.05 mM PLP. The samples were incubated at 30ºC. Samples were taken at 30 minutes, on the hour, at 3 hours and during the

10 night (19 hours). Table 25 shows the averaged results of the total monatin produced at each time interval and the% of (R, R) -monatin produced, as determined by the peak areas of the reverse phase. In column 3 of Table 25, an additional FDAA derivatization analysis by LC / MS / MS was performed as described in Example 1, for some of the reactions and these results are shown in parentheses.

Table 25: Total monatin produced from D-tryptophan and% R, R

Aldolasa (hours)

Total monatin (ppm) % of (R, R) -monatin

SEQ ID NO: 28 (0.5)

16 99.1

SEQ ID NO: 28 (1)

53.2 99.2 (99.0)

SEQ ID NO: 28 (3)

207.8 98.6 (98.1)

SEQ ID NO: 28 (19)

544.9 95.3 (93.2)

SEQ ID NO: 44 (0.5)

46.2 88.0

SEQ ID NO: 44 (1)

92.5 86.8

SEQ ID NO: 44 (3)

319.7 76.4

SEQ ID NO: 44 (19)

762.9 67.1

SEQ ID NO: 54 (0.5)

35.3 96.2

SEQ ID NO: 54 (1)

82.7 96.1

SEQ ID NO: 54 (3)

280.1 92.9

SEQ ID NO: 54 (19)

715.3 77.1

SEQ ID NO: 74 (0.5)

51.1 92.6

SEQ ID NO: 74 (1)

89.3 94.3

SEQ ID NO: 74 (3)

269.5 89.9

SEQ ID NO: 74 (19)

701.9 76.2

SEQ ID NO: 50 (0.5)

55.9 96.7

SEQ ID NO: 50 (1)

96.5 96.2

SEQ ID NO: 50 (3)

272.2 95.6

SEQ ID NO: 50 (19)

645.8 88.5

SEQ ID NO: 162 (0.5)

37.3 95.7

SEQ ID NO: 162 (1)

75.0 97.1

SEQ ID NO: 162 (3)

261.0 95.9

SEQ ID NO: 162 (19)

633.1 87.0

SEQ ID NO: 276 (0.5)

37.8 98.8

SEQ ID NO: 276 (1)

71.2 99.3 (99.5)

SEQ ID NO: 276 (3)

245.2 99.0 (99.0)

SEQ ID NO: 276 (19)

585.4 96.7 (96.1)

SEQ ID NO: 244 (0.5)

30.2 97.7

SEQ ID NO: 244 (1)

46.4 98.3 (99.2)

SEQ ID NO: 244 (3)

165 98.4 (98.7)

SEQ ID NO: 244 (19)

572.5 95.6 (93.7)

SEQ ID NO: 228 (0.5)

52 95.0

SEQ ID NO: 228 (1)

81.7 96.5

SEQ ID NO: 228 (3)

251 95.9

SEQ ID NO: 228 (19)

723 87.1

without aldolase (0.5)

0

aldolase-free (1)

0

aldolase-free (3)

0.6 58.3

aldolase-free (19)

6.5 61.5

The aldolase from SEQ ID NO: 276 maintained a high level of activity, as well as the highest stereospecificity for the production of (R, R) -monatin. Storage of this enzyme in a buffer with the omission of sodium chloride appears to reduce the level of precipitation noted above. The magnesium concentration in the buffer

Storage does not appear to have an impact on the level of precipitation.

The aldolases of SEQ ID NO: 276, SEQ ID NO: 28 and SEQ ID NO: 244 all demonstrated high activity and high stereoselectivity for the production of (R, R) -monatin. These three enzymes were prepared as described in Example 23 and anaerobically tested as described in Example 24, using 10 mL serum containers. 7 mL assays were performed using 0.05 mg / mL of each aldolase (purified) and 2 10 mg / mL of purified B. sphaericus D-aminotransferase prepared as described in Example 24. The activity of each aldolase in monatin production from D-tryptophan was compared to aldolase HMG from

S. meliloti prepared as described in Example 27. Total monatin was estimated using the LC-OPA procedure described in Example 1. The percentage of (R, R) -monatin formed was determined using the procedure of FDAA derivatization described in Example 1. Results are shown at

15 continued in table 26.

Table 26

Aldolasa

Time (hours) Monatina (g / L) % R, R formed

S. meliloti

2. 3 3.9 82.0

SEQ ID NO: 28

2. 3 4.0 84.6

SEQ ID NO: 276

2. 3 4.0 95.7

SEQ ID NO: 244

2. 3 3.7 88.8

S. meliloti

47 4.5 76.2

SEQ ID NO: 28

47 4.3 84.6

SEQ ID NO: 276

47 4.3 93.2

SEQ ID NO: 244

47 4.5 85.2

These results demonstrate that the aldolase of SEQ ID NO: 276 also produces high levels of (R, R) monatin in higher volume anaerobic reactions.

Example 23

Expression and purification of aldolase from SEQ ID NO: 276

Cloning of the E. coli BL21 (DE3) pLysS receptor that has the aldolase gene listed as SEQ ID NO: 275 in plasmid pET28b is described above in Example 22.

The aldolase from SEQ ID NO: 276 with an amino-terminal HIS6 purification marker was produced using the Overnight Express II system from Biosciences EMD (Novagen, Madison, WI) (solutions 1 to 6) containing 50 µg / mL of kanamycin in shake flasks. This expression system induces the expression of IPTG-induced systems without the need to control cell growth. After inoculation of 200 mL aliquots of the medium (in 1 L flasks) from the liquid cultures or plates from the aldolase construct, the cultures were incubated at 30 ° C overnight with shaking at 225 rpm. When OD600, n, reached a minimum of 6, cells were harvested by centrifugation and washed once with buffer.

To prepare the cell free extract containing aldolase, cells were suspended in 3 to 4 volumes of 100 mM potassium phosphate, pH 7.8 and then mixed by using a Microfluidics homogenizer (Microfluidics, Newton, MA ) (3 passes at 18,000 psi), with maintenance of the suspension temperature below 15ºC. Alternatively, the cell-free extract was prepared using EMD Biosciences' BugBuster® Extraction Reagent (primary amine free) (Novagen, Madison, WI) containing 1 µL / mL Benzonasa® Nuclease, 5! L / mL mix Protease Inhibitor II mixture and 0.033 µL / mL rLisozyme ™ according to manufacturer's protocol. All subsequent purification steps were carried out at 4 ° C. The cell suspension was centrifuged for 20-30 minutes at 15,000-20,000 × g to remove cell debris. A 20 to 25 mL aliquot of the cell free extract was applied to a 45 mL column of GE Healthcare Chelating Sepharose ™ Rapid Flow Resin (Nickel (II) form) (GE Healthcare, Piscataway, NJ) that had been previously equilibrated with 100 mM potassium phosphate containing 200 mM sodium chloride. To generate nickel from the resin, the resin was washed with 150 mL of 200mM nickel (II) sulfate hexahydrate and then with 150 mL of distilled water. After loading the sample, the column was washed / eluted with 150 mL of the equilibration buffer containing 25 mM imidazole, 150 mL of the equilibration buffer containing 50 mM imidazole and 150 mL of the equilibration buffer containing 500 mM of imidazole. The HIS6-labeled protein was eluted in the last wash. The 500 mM imidazole wash was concentrated with Millipore / Amicon Centricon Plus-70 Filter (MWCO 10kDa) (Millipore, Billerica, MA) to 15-20 mL according to the manufacturer's instructions. Imidazole and sodium chloride were removed by passage through the GE Healthcare PDIO (GE Healthcare, Piscataway, NJ) disposable columns (2.5 mL sample per column) pre-equilibrated with 100 mM potassium phosphate, pH 7.8. The purified aldolase was eluted with 3.5 mL per column of the same buffer.

The protein concentration of each fraction was determined using the Pierce BCA Assay Kit (Pierce, Rockford, IL) using BSA as the protein standard. The purity of each fraction and the level of expression in the cell-free extract were determined by using a Bio-Rad Experion microcapillary chip system (Bio-Rad, Hercules, CA) or by using SDS gradient gels. -polyacrylamide from 4 to 15% of Bio-Rad in a Mini PROTEAN® 3 cellular device (Bio-Rad, Hercules, CA). Protein was visualized on polyacrylamide gels using Bio-Rad Bio-Safe Coomassie G-250 staining (Bio-Rad, Hercules, CA) and stained with water. Typically, this procedure produces approximately 50 mg of enzyme from 400 mL of culture overnight that is 85 to 95% pure, judging by the Experion software. Aliquots (1 to 5 mL) of the purified enzyme were stored at −80 ° C until use. Preparation of the enzyme in this form reduced the level of precipitation of the enzyme noted above. The presence of magnesium in the storage buffer had no effect on the level of precipitation.

Example 24

Production of (R, R) -monatin by using aldolase of SEQ ID NO: 276: Optimization of reaction conditions

The Bacillus sphaericus D-alanine aminotransferase (ATCC strain 10208) cloned in Example 7 was purified as the HIS6-labeled protein as described below using the EMD Biosciences Overnight Express System II (Novagen, Madison, WI) to its cultivation and induction. The EMD Biosciences Overnight Express System II (solutions 1 to 6) (Novagen, Madison, WI) contained 50 µg / mL kanamycin in shake flasks. This expression system induces the expression of IPTG-induced systems without the need to control cell growth. After inoculation of 200 mL aliquots of the medium (in 1 L flasks) from the liquid cultures or plates from the aminotransferase construct, the cultures were incubated at

30 ° C overnight with stirring at 225 rpm. When the OD600nm reached a minimum of 6, the cells were harvested by centrifugation and washed once with buffer.

To prepare the cell-free extract containing D-alanine aminotransferase, the cells were suspended in 3 to 4 volumes of 100 mM potassium phosphate, pH 7.8, containing 50 µM pyridoxal phosphate (PLP), and then they were mixed using a Microfluidics homogenizer (Microfluidics, Newton, MA) (3 passes at 18,000 psi), keeping the temperature of the suspension below 15 ° C. Alternatively, the cell-free extract was prepared using EMD Biosciences' BugBuster® Extraction Reagent (primary amine free) (Novagen, Madison, WI) containing 1 µL / mL Benzonasa® Nuclease, 5! L / mL mix Protease Inhibitor II mixture and 0.033 µL / mL rLisozyme ™ according to manufacturer's protocol. All subsequent purification steps were carried out at 4 ° C. The cell extract was centrifuged for 20-30 minutes at 15,000 xg to extract cell debris. A 20 to 25 mL aliquot of the cell free extract was applied to a 45 mL column of GE Healthcare Chelating Sepharose ™ Rapid Flow Resin (Nickel (II) form) (GE Healthcare, Piscataway, NJ) that had been previously equilibrated with 100 mM potassium phosphate containing 200 mM sodium chloride and 50 µM PLP. To generate nickel from the resin, the resin was washed with 150 mL of 200mM nickel (II) sulfate hexahydrate and then with 150 mL of distilled water. After loading the sample, the column was washed / eluted with 150 mL of the equilibration buffer containing 25 mM imidazole, 150 mL of the equilibration buffer containing 50 mM imidazole and 150 mL of the equilibration buffer containing 500 mM of imidazole. The HIS6-labeled protein was eluted in the last wash. The 500 mM imidazole wash was concentrated with Millipore / Amicon Centricon Plus-70 Filter (MWCO 10 kDa) (Millipore, Billerica, MA) to 15 to 20 mL according to the manufacturer's instructions. Imidazole and sodium chloride were removed by passage through the GE Healthcare PDIO (GE Healthcare, Piscataway, NJ) disposable columns (2.5 mL sample per column) pre-equilibrated with 100 mM potassium phosphate, pH 7.8, containing 0.5 µM PLP. The purified aminotransferase was eluted with 3.5 mL per column of the same buffer.

The concentration of each fraction was determined using the Pierce BCA Assay Kit (Pierce, Rockford, IL) using BSA as the protein standard. The purity of each fraction and the level of expression in the cell-free extract were determined by using a Bio-Rad Experion microcapillary chip system (Bio-Rad, Hercules, CA) or by using SDS gradient gels. -4 to 15% polyacrylamide of Bio-Rad (Bio-Rad, Hercules, CA) in a Mini PROTEAN® 3 cellular apparatus. The protein was visualized in polyacrylamide gels using Bio-Coomassie G-250 staining. Rad Bio-Safe (Bio-Rad, Hercules, CA) and stained with water. Typically, this procedure produces approximately 50 mg of enzyme from 200 mL of culture overnight that is 85 to 90% pure, judging by the Experion software and from analysis of SDS-PAGE gels. Aliquots (1 to 5 mL) of the purified enzyme were stored at −80 ° C until use.

The aldolase from SEQ ID NO: 276 (cloned in Example 22) was purified as the HIS6-labeled protein as described in Example 23.

The preferred metal cofactor for aldolase of SEQ ID NO: 276 was determined by analysis of a variety of divalent metals. Reactions were carried out anaerobically in 10 mL serum bottles with 7 mL of final volumes. The bulk solution consisting of 100 mM potassium phosphate (pH 7.8), 200 mM sodium pyruvate, 0.05 mM PLP, and 0.01% Tween 80 (v / v), was prepared at a final volume of 48.8 mL and was sprayed with nitrogen for 30 minutes. D-tryptophan (143 mg; final concentration of 100 mM) was placed in seven 10 mL serum containers. 0.014 mL of a 2 M bulk solution of a divalent metal cation, prepared from chloride salt (final concentration of 4 mM), was added to each of the vessels. For the negative control, 0.014 mL of dH2O was added. The serum containers were capped with rubber septa and sparged with nitrogen using 16 to 18 sterile needles. Under anaerobic conditions, 5.625 mL of the anaerobic bulk solution was added to each vial of anaerobic serum. Subsequently, D-alanine aminotransferase from

B. sphaericus and aldolase from SEQ ID NO: 276 were added anaerobically to each vial of serum at a final concentration of 2 mg / mL and 0.05 mg / mL, respectively. The solutions were incubated at room temperature with gentle mixing for 18 hours. The final monatin was analyzed according to the procedures described in Example 1 using the post-column liquid chromatography fluorescent amino acid detection procedure. (Table 27).

Table 27

Metal cofactor

Final monatina (mM) at 6 p.m.

None (negative control)

1.7

Magnesium

10.6

Manganese

10.0

Metal cofactor

Final monatina (mM) at 6 p.m.

Cobalt

6.7

Zinc

4.9

Nickel

1.5

Calcium

 0.7

The reaction conditions for aldolase of SEQ ID NO: 276 were further investigated with a two-level fractional factorial experiment designed using the Design Expert statistical software.

7.0.0 (Stat-Ease, Inc .; Minneapolis, MN). The analysis design consisted of a single block of five factors to two

5 levels with four central points (20 executions in total). The five factors to be optimized were the metal cofactor concentration, the reaction pH, the Tween® 80 concentration, the pyruvate to tryptophan ratio and the aldolase concentration (Table 28).

Conical polypropylene tubes (14 mL) containing 143 mg of D-tryptophan were deoxygenated in an anaerobic chamber (Coy Laboratory Products, Inc; Grass Lake, MI) overnight. 2M 10 MgCl2 bulk solutions; 1M potassium phosphate at pH 7.0, 7.75 and 8.5; Tween® 80 (v / v) 10%; 2M sodium pyruvate and 10mM PLP (pyridoxal 5'-phosphate) were prepared in degassed water and equilibrated in the anaerobic chamber overnight. Bulk solutions of B. sphaericus D-alanine aminotransferase and aldolase from SEQ ID NO: 276 were immediately thawed on ice in an anaerobic chamber. Bulk solutions were added to the 14 mL conical tubes containing the D-tryptophan to obtain the concentrations 15 determined by the statistical design (Table 28). Degassed water was added to each tube to bring the final volume, along with enzyme additions, to 7.0 mL. The tubes were incubated at room temperature in an anaerobic chamber with gentle mixing for 24 hours. Monatin concentration and isomeric purity were analyzed according to the procedures described in Example 1, using the post-column liquid chromatography fluorescent amino acid detection procedure and the multiple reaction control

20 by LC / MS / MS for the determination of the distribution of monatin stereoisomers in the in vitro and in vivo reaction procedure (FDAA derivatization procedure).

Table 28

Execution #

standard # Block Mg (mM) pH TweenD® (%) Pir: Trp Aldolase from SEQ ID NO: 276 (mg / mL)

twenty

one Block 1 5.00 7.75 0.01 2.00 0.05

8

2 Block 1 9.00 8.50 0.02 1.00 0.01

3

3

Block 1 1.00 8.50 0.00 1.00 0.01

16

4 Block 1 9.00 8.50 0.02 3.00 0.09

7

5 Block 1 1.00 8.50 0.02 1.00 0.09

12

6 Block 1 9.00 8.50 0.00 3.00 0.01

6

7 Block 1 9.00 7.00 0.02 1.00 0.09

2

8 Block 1 9.00 7.00 0.00 1.00 0.01

fifteen

9 Block 1 1.00 8.50 0.02 3.00 0.01

4

10 Block 1 9.00 8.50 0.00 1.00 0.09

5

eleven Block 1 1.00 7.00 0.02 1.00 0.01

one

12 Block 1 1.00 7.00 0.00 1.00 0.09

13

13

Block 1 1.00 7.00 0.02 3.00 0.09

Execution #

standard # Block Mg (mM) pH TweenD® (%) Pir: Trp Aldolase from SEQ ID NO: 276 (mg / mL)

14

14

Block 1 9.00 7.00 0.02 3.00 0.01

17

fifteen Block 1 5.00 7.75 0.01 2.00 0.05

eleven

16 Block 1 1.00 8.50 0.00 3.00 0.09

18

17 Block 1 5.00 7.75 0.01 2.00 0.05

9

18 Block 1 1.00 7.00 0.00 3.00 0.01

19

19

Block 1 5.00 7.75 0.01 2.00 0.05

10

twenty Block 1 9.00 7.00 0.00 3.00 0.09

Statistical analysis of the data indicated that the reaction pH, pyruvate: tryptophan ratio, and aldolase concentration were significant factors affecting monatin titration, isomeric purity, and carbon production. A convenience graph was generated by using the Design Expert software in which

5 factors were varied to maximize the goals of higher monatin titration and higher isomeric purity under pyruvate excess conditions. The reaction conditions indicated as optimal were 1 mM MgCl2, pH> 8.0, Tween® 80 0.01% (v / v) and 0.01 mg / mL aldolase from SEQ ID NO: 276. This is a 5-fold reduction in the typical amount of aldolase used, as well as a 4-fold reduction in the amount of divalent metal typically used.

Additional experiments were performed to determine the optimal pH range for the reaction process. Bulk 1M EPPS buffer solutions were prepared in pH unit increments of 0.2 between pH 7.0 and 9.0. These solutions were degassed and equilibrated in the anaerobic chamber overnight. Polypropylene tubes (14 mL) containing 143 mg of D-tryptophan were deoxygenated in an anaerobic chamber overnight. Bulk solutions of 2M MgCl2, Tween® 80 10% (v / v), 2M Sodium Pyruvate and 10mM

15 PLP were prepared in degassed water and equilibrated in the anaerobic chamber. The B. sphaericus D-alanine aminotransferase and aldolase preparations of SEQ ID NO: 276 were thawed on ice and immediately used in the anaerobic chamber. Bulk solutions were added to the 14 mL conical tubes to provide a final concentration of 100 mM EPPS, 200 mM pyruvate, 100 mM tryptophan, 1 mM MgCl2, 0.01% Tween® 80 (v / v), 0.05 mM PLP, 2 mg / mL D-alanine aminotransferase from B. sphaericus and 0.01

20 mg / mL of aldolase from SEQ ID NO: 276 in a total volume of 7 mL per tube. Reactions were incubated at room temperature in an anaerobic chamber with gentle shaking for 22 hours. Samples were removed and analyzed to verify monatin as described in Example 1 using the LC / MS / MS Multiple Reaction Control Procedure (Table 29).

Table 29

reaction pH

Monatina (mM) at 22 hours

7.0

5.8

7.2

9.9

7.8

14.0

8.0

14.2

8.2

14.3

8.4

12.6

8.6

12.3

8.8

10.8

9.0

11.1

Results indicated that monatin formation increased with increasing pH between 7.0 and 8.0. Monatin formation peaked in the pH range of 8.0 and 8.2 and decreased at a pH greater than 8.4. Furthermore, the monomer isomeric purity decreased at a pH greater than 8.4.

Further reaction optimization was performed using aldolase SEQ ID NO: 276 (0.01 mg / mL), 2 mg / mL T243N 4978 DAT (unlabelled from Example 26), 1 mM MgCl2 , 200 mM sodium pyruvate, 0.05 mM PLP, 0.01% Tween-80 and 100 mM D-tryptophan at pH 8.5. The cells used to produce aldolase and D-aminotransferase were opened in 50 mM EPPS, pH 8.4, using a Microfluidics homogenizer (Microfluidics, Newton, MA) (3 passes at 20,000 psi). Cell debris was removed by centrifugation (20,000 × g for 30 min) and the clarified cell extracts were used in the enzyme reactions. No additional buffer was used, but the reaction mixtures were adjusted to pH 8.5 by using sodium hydroxide and rinsed with nitrogen before addition of the enzyme. Reactions of 250 mL in 0.7 L were carried out, six for stirred fermenters (Infors AG, Bottmingen, Switzerland) at 30 ° C using nitrogen in the air space. Potassium phosphate was added at a final concentration of 0, 5, 10, 20, and 50 mM. The addition of 5 to 10 mM phosphate was found to be optimal, which produced 3.5 g / L monatin (quantified by LC / MS / MS as described in example 1).

Example 25 (not claimed)

Cloning of a new Bacillus D-amino acid aminotransferase

Bacillus D-amino acid aminotransferase ("DAAT" or "DAT") (EC 2.6.1.21, also known as Dalanine aminotransferase or D-aspartate aminotransferase) was produced recombinantly. This aminotransferase was used in aldolase-coupled assays for the production of (R, R) -monatin. This aminotransferase enzyme is homologous to the D-aminotransferases previously described for the production of monatin (published US application No. 20040063175 and published US application No. 20050282260). ATCC 4978 — Bacillus sphaericus originally deposited as Bacillus rotans — was requested from ATCC and used to prepare genomic DNA. Degenerate primers in the conservation regions of known Bacillus D-aminotransferase protein sequences were designed and used for amplification of the polymerase chain reaction ("PCR") of the gene sequence of the internal DAAT of the ATCC strain mentioned above. The genomic walk was performed using the BD GenomeWalker ™ Universal Kit (Clontech, Mountain View, CA). Sequence analyzes (Agencourt BioScience Corporation, Beverly, MA) verified a full-length coding sequence for the ATAT 4978 DAAT gene. The DNA sequence of the ATCC 4978 DAAT gene is SEQ ID NO: 357 and is shown then. The gene in SEQ ID NO: 357 can be amplified through standard PCR protocols and cloned using recombinant DNA techniques. The gene of SEQ ID NO: 357 can also be reconstructed by any method known to the person skilled in the art, such as the PCR assembly procedures known to the person skilled in the art.

The DAAT ATCC 4978 DNA sequence is: (SEQ ID NO: 357)

The amino acid sequence of the ATAT 4978 DAAT gene as encoded by the DNA sequence above is SEQ ID NO: 358 and is shown below:

(SEQ ID NO: 358)

The new D-aminotransferase obtained from the ATCC 4978 strain has a protein sequence that exhibits different changes of amino acid residues when compared to the published D-aminotransferase sequence of ATCC 10208 from B. sphaericus. The DAAT of ATCC 4978 has only 72% identity with the

DAAT of B. sphaericus (ATCC 10208). While this strain is listed as B. sphaericus in the ATCC, it was deposited as B. rotans. Based on the sequence alignments and the prominent differences between this new DAAT and the B. sphaericus DAAT, several candidate residues were identified that can be evaluated for their role (individually or in combination) in the increased DAAT activity for biosynthesis of (R, R) -monatin, in these, as well as other DAAT sequences.

Example 26 (not claimed)

Characterization of ATCC 4978 D-Aminotransferase Mutants

Experimental summary

The new D-aminotransferase gene (described in Example 25) from Bacillus strain ATCC 4978 was mutagenized using site-directed procedures. Mutant genes were expressed and tested to verify

20 activities of interest to the monatin production pathways, especially when coupled to one or more aldolases.

In addition to the ideas listed in Example 16 for the purposes of site-directed mutagenesis, other ideas were developed for the actual anchoring of R-MP to the active site of the YM-1 crystal structure, the

Primary amino acid sequence alignments were used to determine whether the 4978 protein aminotransferase was likely to have structural features in that region. The following additional mutations were expected to be beneficial (using the amino acid numbering of aminotransferase 4978). Mutagenesis of alanine 153 to arginine was thought to stabilize the second carboxyl group in the substrate (R-MP). This change is likely to increase steric hindrance, to compensate for this, serine residues at positions 181 and 182 were changed to alanine or glycine. We also hypothesized that an arginine could be introduced at position 180, 181, or 182 and one or more of the other serine residues converted to alanine or glycine to make room for the more bulky arginine side chain. The phenylalanine at amino acid 200 is spatially close to the place where R-MP is predicted to anchor at the active site and there is wide variability in this residue among D-aminotransferases that catalyze monatin transamination quite well. Modifications of amino acids at this position were thought to be useful. The mutation of leucine 151 to phenylalanine was predicted to potentially improve interactions with the indole ring of the substrate.

Based on the literature, it was hypothesized that the mutation of threonine 243 to aspargine can improve the selectivity of R-MP for transamination reactions. Likewise, it was thought that mutagenesis of aspargine 100 to alanine may improve the specific activity of the enzyme for monatin transamination reactions (Ro, et al., FEBS Lett 398: 141-145, (1996); Sugio, S, et al., Biochemistry 34: 9661-9669, (1995); EP1580268).

Lee et al. characterized mutants from region 141 to 144 (loop) and found that D-aminotransferases with EYcY instead of LRcD (which is native to our protein) tend to have a lower Km for dicarboxylic acid substrates. (Lee SG, Hong SP, Song JJ, Kim SJ, Kwak MS, Sung M H. Functional and structural characterization of thermostable D-amino acid aminotransferases from Geobacillus spp. Appl Environ Microbiol. 2006 February; 72 (2): 1588-94 ). Because MP is a dicarboxylic acid substrate, similar to alpha-keto glutarate, and MP concentrations are quite low in a monatin production reaction mixture, a lower Km could potentially aid the activity of a mutant DAT to monatin production.

The procedures for creating the D-aminotransferase 4978 mutants are described below, as well as analyzing the results with the use of these mutants.

Mutagenesis

Mutagenesis primers were designed according to the suggestions listed in the Stratagene Multi-Change Kit (Stratagene, La Jolla, CA). The primers were 5'-phosphorylated. Mutagenesis was performed using the Stratagene Multi-Change Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The templates used for mutagenesis were the DAT 4978 constructs of pET30 (unlabeled) or pET28 (labeled) described in Example 25. The primer sequences are listed below in Table 30:

Table 30

Mutant name

Amino acid change Primer

4978-22

T243N GTGATTGTTTCATCAACGAATTCAGAAGTAACGCC (SEQ ID NO: 359)

10

T243R GTGATTGTTTCATCAACGCGTTCAGAAGTAACGCC (SEQ ID NO: 360)

7

T243S GTGATTGTTTCATCAACGAGTTCAGAAGTAACGCC (SEQ ID NO: 361)

19

T243A GTGATTGTTTCATCAACGGCTTCAGAAGTAACGCC (SEQ ID NO: 362)

fifteen

N100A GTGCAGGCCCTCGTGCTCATATTTTCCCTGG (SEQ ID NO: 363)

B

T243Q GAAGTGATTGTTTCATCAACGCAGTCAGAAGTAACCGCCAATTAT C (SEQ ID NO: 364)

2

T243N / N100 the above primers are used together

E.coli XL10-Gold cells (Stratagene, La Jolla, CA) were transformed and the resulting purified plasmid preparations were sequenced to verify that the correct mutations were incorporated.

Expression and essay

Plasmid DNA preparations containing either the correct mutants or wild-type DAT 4978 were transformed into the E. coli expression host BL21 (DE3) (Novagen, Madison, WI). Cultures were grown using the protocols described above, plasmids were isolated using the Qiagen miniprep kit (Qiagen, Valencia, CA) and analyzed by restrictive digestion, as described above, to confirm the presence of a insertion.

DAAT gene induction was typically performed in LB medium containing kanamycin (50 µg / mL). Cells were grown at an OD600nm of 0.4 to 0.8 at 37 ° C, induced with 0.1mM IPTG (isopropyl thiogalactoside) and sampled 3 to 4 hours after induction.

Cell extracts were prepared with BugBuster reagent and benzase nuclease (Novagen, Madison, WI). 1 ml assays were performed at 30 ° C with gentle shaking containing 10.2 mg D-tryptophan, 0.05 mM PLP, 4 mM MgCl2, 100 mM potassium phosphate buffer, pH 7.5, approximately 50 µg aldolase, 200 mM pyruvate and 0.150 to 0.5 mg / mL D-aminotransferase provided as cell extracts. All protein assays were performed using the Bio-Rad (Coomassie) Total Protein Kit (Bio-Rad, Hercules, CA) or Pierce's BCA Kit (Pierce, Rockford, IL) and percent expression D-aminotransferase was estimated by SDS-PAGE or the Bio-Rad Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA). Samples were taken at 3 hours and overnight.

The R-specific aldolase of SEQ ID NO: 28 was used in assays with approximately 0.150 mg / mL Daminotransferase.

The first tests showed that the following mutants (unlabeled) showed transamination activity (in order from highest to lowest): T243N, T243S, T243N / N100A, N100A. It was noted that T243N seemed to increase the stereo-purity of the (R, R) -monatin produced. The assays were repeated using ProA aldolase purified from Comamonas testosteroni (100 µg / ml) and 0.50 mg / ml D-aminotransferase mutants (unlabelled, provided as cell extract). Samples were taken at 2 hours and overnight. Results for active proteins are shown below, duplicate results were averaged. The% of (R, R) -monatin was determined by peak area on reverse phase HPLC and then measured by using the FDAA derivatization procedure described in Example 1. In column 3 of Table 31, Additional FDAA derivatization analysis by LC / MS / MS was performed as described in Example 1 for some of the reactions and these results are shown in parentheses. Only R, R and (S, R) -monatin are produced from Dipryptophan. The T243R mutant did not appear to produce monatin under the conditions tested and the T243A mutant produced very low levels of monatin.

Table 31

Unmarked enzyme (time-hour or overnight)

Total monatin (ppm) % R, R

wild guy 4978 (2 hours)

4.7 41.6

wild guy 4978 (overnight)

43.2 35.1 (30.9)

T243S (2 hours)

55.0 37.4 (21.7)

T243S (overnight)

97.7 35.5 (29.8)

T243N (2 hours)

73.2 86.7 (88.3)

T243N (overnight)

120.9 86.3 (86.1)

N100A (2 hours)

12.0 40.8

N100A (overnight)

22.3 41

T243A (2 hours)

0.8 ~ 100

Although the tests were performed with the estimation of the percentage of D-aminotransferase by using the Bio-Rad Experion software, it is clear that the T243S and T243N mutants show a higher activity compared to the wild type enzyme. The T243N mutant further provided an additional benefit of the dramatic increase in% of (R, R) -monatin formed. This enzyme has a higher preference for R-MP compared to S-MP in transamination reactions. The N100A mutant did not increase activity on its own or in combination with T243N, contrary to what was suggested in the literature. The unlabelled DAT 4978 site-directed V34A mutant was also created using similar procedures as described above. The site-directed V34A mutant was found to have significantly less activity than the wild-type enzyme under the conditions tested.

Another point of interest in the initial assays was that the wild-type enzyme appeared to have increased activity when produced with an N-terminal His marker. Subsequent mutagenesis was performed in the labeled version of the gene. In addition, the most promising mutants above were subcloned into pET28b that had an N-terminus His marker. These were purified using Novagen HIS-bind columns and the manufacturer's protocol with the recommended buffers (Novagen, Madison, WI). The buffer of the eluent fractions was exchanged, using GE Healthcare PD10 columns (GE Healthcare, Piscataway, NJ), with the buffer used in the assays.

1 ml assays were performed with purified D-aminotransferase (0.5 mg / ml) and purified R-specific aldolase from SEQ ID NO: 276 (50 µg / ml) at 30 ° C with slight agitation and containing 10.2 mg D-tryptophan, 0.05 mM PLP, 200 mM pyruvate, 4 mM MgCl2 and 100 mM potassium phosphate buffer, pH 7.5. Duplicate samples were incubated for 2 hours and overnight. As a positive control, the DAT of Bacillus sphaericus (cloned in Example 7) was used in the same tests. The results are presented in table 32:

Table 32

Enzyme - marked (time: hour or overnight)

Total monatin (ppm) % R, R

wild guy 4978 (2 hours)

43 98.4

wild guy 4978 (overnight)

96.7 98.3 (95.9)

T243N (2 hours)

197.5 100

T243N (overnight)

301.2 99.9 (99.6)

B. sphaericus DAT (2 hours)

58.2 99.7

B. sphaericus DAT (overnight)

221.7 98.7 (96.6)

T243Q (2 hours)

7.1 100

T243Q (overnight)

12.4 98.8

The above data showed that the T243N mutant clearly produces the highest amount of monatin at 2 hours. As time passes, the ratio of the positive control of the T243N mutant to the DAT of

B. sphaericus is reduced. This result suggests that the T243N mutant is not as stable during the monatin reaction as the B. sphaericus DAT. When analyzed under similar conditions, the enzyme T243S (branched, purified) exhibited activity levels similar to those of the T243N mutant; however, the percentage of (R, R) monatin produced was lower (97.2% in both 2 hours and during the night). The T243N / N100A mutant has less activity than the T243N mutant. However, both T243S and T243N / N100A exhibited higher activity than wild type DAT 4978.

Transamination assays were performed to determine what reaction rates were improved by using the T243N mutant instead of the B. sphaericus DAT. 0.5 ml tests were carried out at 30 ° C and 1 hour, 2 hours and 5 hours were taken as time intervals. The assays contained 25 mM monatin or Dtryptophan, 25 mM pyruvate, 100 mM potassium phosphate, pH 7.5, 50 µM PLP and 0.1 mg D-aminotransferase (labeled, purified). In the case where less than 100 µg of DAT was used, the amount of alanine was normalized to 100 µg of D-aminotransferase. The samples were treated with formic acid and analyzed by LC-OPA to verify the presence of the co-product, alanine. The results are shown in Tables 33 and 34.

Table 33: Transamination activity with (R, R) -monatin as substrate

Enzyme

D-Alanine (mM)

DAT 4978 wild type (2 hours)

0.54

DAT 4978 wild type (5 hours)

1.11

T243N / N100A (2 hours)

1.32

T243N / N100A (5 hours)

2.78

T243S (2 hours)

1.5

T243S (5 hours)

2.61

T243N (2 hours)

1.26

T243N (5 hours)

2.65

B. sphaericus DAT (2 hours)

0.97

B. sphaericus DAT (5 hours)

2.2

Table 34: Transamination activity with D-tryptophan as substrate

Enzyme

D-Alanine (mM)

DAT 4978 wild type (1 hour)

4.55

DAT 4978 wild type (2 hours)

8.47

T243N / N100A (1 hour)

8.52

T243N / N100A (2 hours)

12.67

T243S (1 hour)

4.89

T243S (2 hours)

8.1

T243N (1 hour)

7.19

T243N (2 hours)

10.83

B. sphaericus DAT (1 hour)

8.7

B. sphaericus DAT (2 hours)

12.54

For D-tryptophan reactions, the results show that some of the enzymes reached equilibrium within 2 hours. The (R, R) -monatin reactions clearly limit the rate and improvements to this activity have a greater impact on the monatin production rates of D-tryptophan.

Additional assays were performed to examine the stability of the D24 4978 T243N mutant.

The wild guy also loses activity over time. Example 27 describes procedures to improve the stability of the T243N mutant of D-aminotransferase. When the freshly labeled and unlabelled versions of the T243N mutant were prepared and compared for activity, the unlabelled version was found to have better temporal stability, making it a better version of the enzyme for use in reactions of monatina production.

Other mutants of DAT 4978 were prepared by procedures commonly known to those skilled in the art. However, all these mutations resulted in a protein that was insoluble under the conditions in which they were prepared and therefore could not be analyzed to verify its activity. The mutations that resulted in insoluble proteins were:

S180 A / S181A / S182R; L151F; V34G; S181R; A153R / S181A / S182A; A153R / S182A; A153R / S182G; S180R / S181A / S182G; S180R / S181A / S182A; S180R / S181G / S182G; S180G / S181R / S182G; and S180A / S181R / S182A. Additional mutagenesis To create the F200M mutant from DAT 4978, the wild type DAT 4978 open reading frame from the example

25 (labeled) was amplified with primers 73 and 80 (below) and PfuTurbo DNA polymerase (Stratagene,

La Jolla, CA) and cloned into pCRII-Blunt (Invitrogen, Carlsbad, CA). Their sequence was verified (Agencourt, Beverly,

MA). The 5 'and 3' regions were amplified using primers 80 and 96 and 99 and 103, respectively. The

Amplified DNA was then gel purified using Qiagen QIAquick Gel Extraction Kit

(Qiagen, Valencia, CA). Amplified DNA was again subjected to PCR using primers 80 and

99. Amplified DNA was gel purified as described above and cloned into pCRII Blunt and its sequence verified. The DAT open reading frame was subcloned as a restriction enzyme fragment NdeI / XhoI in pET28b.

Table 35

Primer number

Sequence

73

CATATGAGTTATAGCTTATGGAATGACCAAATTGTGAATG (SEQ ID NO: 365)

80

CTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTC (SEQ ID NO: 366)

96

AATATTTATGGAATTAAAGATGGCGTATTATACACACATCCAGCGAATAACATGATCTTAA ATGGTATTACACGTCAAGTAATCATTAAATGTGC (SEQ ID NO: 367)

99

GGCCAGTGAATTGTAATACGACTCACTATAGGGC (SEQ ID NO: 368)

103

CGCCATCTTTAATTCCATAAATATTTGAAGAAGAGCCTTCTG (SEQ ID NO: 369)

The following primers were designed for additional site-directed mutagenesis using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutagenesis was performed using the Stratagene Multi-Change Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The template used for mutagenesis was the DAT 4978 construct of pET28 (labeled) described in Example 25. A double mutant was also created using the F200Y mutant as a template and an additional round of mutagenesis was performed with the T243N primer ( listed above).

Table 36

Mutant

Oligo

141-LRcD144-> EYcY

GCAACATTTGTAGAAGACATTCGTTGGGAATACTGTTACATTAAATCATTAAATTTACTTG GTGCG (SEQ ID NO: 370)

F200Y

 GTATTATACACACATCCAGCGAATAACTACATCTTAAATGGTATTACACGTCAAG (SEQ ID NO: 371)

S244K

GCAATGGATGAAGTGATTGTTTCATCAACGACTAAAGAAGTAACGCCAATTATCGACATA GATG (SEQ ID NO: 372)

243-TS-244> NK

GCAATGGATGAAGTGATTGTTTCATCAACGAATAAAGAAGTAACGCCAATTATCGACATA GATG (SEQ ID NO: 373)

243-TS-244> NR

GCAATGGATGAAGTGATTGTTTCATCAACGAATCGTGAAGTAACGCCAATTATCGACATA GATG (SEQ ID NO: 374)

Mutant coding regions were verified by DNA sequencing (Agencourt). The plasmids to which the sequence was verified were transformed into BL21 (DE3) cells (Novagen, Madison, WI).

Expression and essay

Cultures containing 100 ml of LB with 50 µg / ml kanamycin in a 500 ml baffle flask inoculated with one ml of an overnight culture and grown at 37 ° C at an optical density (at 600nm) of approximately 0, 6. Protein production was induced by IPTG at a final concentration of 1 mM. Cells were incubated at 30 ° C for 4.5 hours after addition of IPTG. Cells were spun and frozen at -80 ° C. Cells (prepared using Novagen's BugBuster reagent (Novagen, Madison, WI) containing 1 µL / mL of benzase nuclease, 5 µL / mL of protease II inhibitor mixture and 0.033 µL / mL of rLisozyme according to the recommended Novagen protocol) they were mixed and analyzed by SDS-PAGE. Mutants (141-LRcD-144-> EYcY) and (243-TS-244-> NR) resulted in insoluble proteins under the conditions in which they were prepared. The 243-TS-244-> NK mutant showed no quantifiable activity under the conditions tested and is probably an enzyme with weak activity compared to the wild type as is the S244K mutant.

His-tagged proteins were purified as follows. The HIS-bind columns (Novagen, Madison, WI) were equilibrated with 10 mL of 100 mM potassium phosphate, pH 7.8, containing 200 mM NaCl and 50 µM PLP. Cell-free extracts were loaded onto the column. The columns were washed with 10mL of equilibration buffer, 10mL of equilibration buffer containing 25mM imidazole and 10mL of equilibration buffer containing 50mM imidazole. Proteins were eluted with 5 ml of equilibration buffer containing 500mM imidazole. Proteins were desalted using PD10 columns that were equilibrated in 100 mM potassium phosphate, pH 7.8 containing 50 µM PLP. Purified proteins were concentrated and quantified using the Bradford Assay (Bio-Rad, Hercules, CA).

D-aminotransferase mutants were analyzed using 500 µg / ml D-aminotransferase, 50 µg / ml aldolase from SEQ ID NO: 276, 4 mM MgCl2, 50 mM potassium phosphate, pH 8,200 mM sodium pyruvate, 0.05 mM PLP and 20.4 mg / ml D-tryptophan for the test conditions. The final volume was 1.25 ml. Samples (200 µl) were taken after 0.5, 1, 2, and 14 hours and frozen until the experiment was completed. The samples were filtered, diluted from 1 to 10, and analyzed by LC / MS / MS as described in Example 1.

Wild type D-aminotransferase 4978 from Example 25 was used as a reference for the relative percentage of activity. Table 37 shows the relative activity of each mutant at each time interval.

Table 37

D-aminotransferase

time (hours) % exercise

4978 wild type

0.5 100

T243N

 0.5 270

F200M

 0.5 fifty

F200Y

 0.5 70

F200M / T243N

 0.5 183

S244K

0.5 4

D-aminotransferase

time (hours) % exercise

4978 wild type

one 100

T243N

 one 289

F200M

 one 55

F200Y

 one 81

F200M / T243N

 one 203

S244K

one 6

4978 wild type

2 100

T243N

 2 266

F200Y

 2 79

F200M / T243N

 2 185

S244K

2 6

4978 wild type

14 100

T243N

 14 254

F200M

 14 56

F200Y

 14 80

F200M / T243N

 14 168

S244K

14 8

Example 27 (not claimed)

T243N was the best mutant of all tested to verify activity in (R, R) -monatin production.

Stabilization of the AT24 4978 strain D-aminotransferase mutant T243N

5 As shown in Example 25, the initial activity of the T243N mutant DAT is significantly higher than for the B. sphaericus DAT, but the activity decreases more rapidly. Additional experiments, using the anaerobic protocol described below, indicated that the initial activity of the T243N mutant DAT was up to 8 times higher than the B. sphaericus DAT, however, the activity decreased rapidly even under anaerobic conditions. The following studies were conducted to try to maintain the highest activity during a

10 extended period of time.

The T243N mutant of the D-aminotransferase of strain 4978 (described in Example 25) and the S. meliloti aldolase HMG were purified as the HIS6-labeled proteins as described below. The aldolase from SEQ ID NO: 276 was purified as described in Example 23.

Purification of DAT from ATCC 4978 (T243N mutant)

The AT24 4978 strain D-aminotransferase mutant T243N with an amino terminal HIS6 purification marker (described in Example 26) was produced using the Overnight Express II system from Biosciences EMD (solutions 1 to 6) ( Novagen, Madison, WI) containing 50 µg / mL kanamycin in shake flasks. This expression system induces the expression of IPTG-induced systems without the need to control cell growth. After inoculation of 200 mL aliquots of the medium (in 1 L flasks) from the cultures

20 liquids or plates of the E. coli BL21 (DE3) receptor bearing the gene for the mutant D-aminotransferase T243N of the ATCC strain 4978 in the plasmid pET28b, the cultures were incubated at 30 ° C overnight with shaking at 225 rpm . When the OD600 reached a minimum of 6, the cells were harvested by centrifugation and washed once with buffer.

The cell free extract was prepared using the EMB Biosciences BugBuster® Extraction Reagent (primary amine free) (Novagen, Madison, WI) containing 1 µL / mL Benzonasa® Nuclease, 5 µL / mL mixture of protease inhibitor set II (Calbiochem — Novabiochem Corp., San Diego, CA) and 0.033 µL / mL rLisozyme ™ (Novagen, Madison, WI) according to the manufacturer's protocol. All subsequent purification steps were carried out at 4 ° C. The cell extract was centrifuged for 20-30 minutes at 15,000 xg to extract cell debris. A 20 to 25 mL aliquot of the cell free extract was applied to a 45 mL column of GE Healthcare Chelating Sepharose ™ Rapid Flow Resin (Nickel (II) form) (GE Healthcare, Piscataway, NJ) that had been previously equilibrated with 100 mM potassium phosphate containing 200 mM sodium chloride and 50 µM PLP. To generate nickel from the resin, the resin was washed with 150 mL of 200mM nickel (II) sulfate hexahydrate and then with 150 mL of distilled water. After loading the sample, the column was washed / eluted with 150 mL of the equilibration buffer containing 25 mM imidazole, 150 mL of the equilibration buffer containing 50 mM imidazole and 150 mL of the equilibration buffer containing 500 mM of imidazole. The HIS6-labeled protein was eluted in the last wash. The 500 mM imidazole wash was concentrated with Millipore / Amicon Centricon Plus-70 Filter (MWCO 10 kDa) (Millipore, Billerica, MA) to 15 to 20 mL according to the manufacturer's instructions. Imidazole and sodium chloride were removed by passage through the GE Healthcare PDIO (GE Healthcare, Piscataway, NJ) disposable columns (2.5 mL sample per column) pre-equilibrated with 100 mM potassium phosphate, pH 7.8, containing 0.5 µM PLP. The purified aminotransferase was eluted with 3.5 mL per column of the same buffer. The concentration of each fraction was determined using the Pierce BCA Assay Kit (Pierce, Rockford, IL) using BSA as the protein standard.

The purity of each fraction and the level of expression in the cell-free extract were determined by using a Bio-Rad Experion microcapillary chip system (Bio-Rad, Hercules, CA) or by using SDS gradient gels. -4 to 15% polyacrylamide of Bio-Rad (Bio-Rad, Hercules, CA) in a Mini PROTEAN® 3 cellular apparatus. The protein was visualized in polyacrylamide gels using Bio-Coomassie G-250 staining. Rad Bio-Safe (Bio-Rad, Hercules, CA) and stained with water. Typically, this procedure produces approximately 20 mg of enzyme from 200 mL of culture overnight that is 85 to 90% pure, judging by the Experion software and from analysis of SDS-PAGE gels. Aliquots (1 to 5 mL) of the purified enzyme were stored at −80 ° C until use.

The aldolase purification of SEQ ID NO: 276 is as described in Example 23.

Purification of S. meliloti HMG aldolase

S. meliloti HMG aldolase with an amino terminal HIS6 purification marker (cloning described in published US application No. 20040063175 and WO 03091396 A2) was produced by induction of cultures grown in Luria-Bertani broth that it contained 50 mg / L kanamycin with 0.2 mM IPTG. After inoculation of 800 mL aliquots of the medium from any of the liquid cultures or plates of the E. coli BL21 (DE3) receptor that has the gene for the S. meliloti HMG aldolase gene in pET30 (Xa / LIC), the Cultures were incubated at 37 ° C with shaking at 225 rpm. When the optical density reached OD600nm from 0.5 to 0.75, IPTG was added and the cultures were incubated at 30 with shaking at 225 rpm for 4 hours. Cells were harvested by centrifugation and washed once with buffer.

To prepare the cell free extract containing S. meliloti aldolase, the cells were suspended in 3 to 4 volumes of 50 mM EPPS (N- (2-hydroxyethyl) piperazine-N '- (3-propanesulfonic acid), pH 8.2 containing 100 mM NaCl and then mixed by using a Microfluidics homogenizer (Microfluidics, Newton, MA) (3 passes at 18,000 psi), with suspension temperature maintained below 15 ° C. Cell suspension was centrifuged for 20-30 minutes at 15,000-20,000 × g to remove cell debris HIS6-labeled protein was purified using HIS-Bind® columns from EMD Biosciences (Novagen, Madison, WI) according with the manufacturer's recommended protocol with one exception: columns were washed with 1: 1 binding buffer: wash buffer solution instead of wash buffer only Elution fraction was concentrated with Millipore filter centrifugation devices / Amicon Centricon Plus15 (MWCO 5k Da) (Millipore, Billerica, MA) at 7 to 10 mL according to the manufacturer's instructions. Imidazole and sodium chloride were removed by passage through GE Healthcare PD1O (GE Healthcare, Piscataway, NJ) disposable columns (2.5 mL sample per column) pre-equilibrated with 50 mM EPPS, pH 8 , 2, containing 100 mM NaCl. The purified aldolase was eluted with 3.5 mL per column of the same buffer. The protein concentration of each fraction was determined using the Pierce BCA Assay Kit (Pierce, Rockford, IL) using BSA as the protein standard.

The purity of each fraction and the level of expression in the cell-free extract were determined by using gels with a gradient of SDS-polyacrylamide from 4 to 15% of Bio-Rad (Bio-Rad, Hercules, CA) in an apparatus Cellular Mini PROTEAN® 3. Protein was visualized on polyacrylamide gels using Bio-Rad Bio-Safe Coomassie G250 stain (Bio-Rad, Hercules, CA) and stained with water. Typically, this procedure produces approximately 15 to 20 mg of the enzyme from 800 mL of culture and is 85 to 95% pure. Aliquots (1 to 5 mL) of the purified enzyme were stored at −80 ° C until use.

Monatin production tests

Polypropylene conical tubes (14 mL) containing 143 mg of D-tryptophan were deoxygenated in an anaerobic chamber overnight. Bulk solutions of 1M EPPS buffer (pH 8.2), 2M MgCl2, 2M sodium pyruvate and 10mM PLP were prepared in degassed water and equilibrated in the anaerobic chamber. 5 Bulk solutions of Tween 80 10% (v / v), Tween 20 1% (v / v), Triton X-100 1% (v / v), 100% acetone, 100% ethanol and 50% glycerol (w / v) were equilibrated in the anaerobic chamber along with 0.7 g of trehalose, inositol, sorbitol and erythritol in 2 mL of microcentrifuge tubes. The purified enzyme preparations were thawed on ice and immediately used in the anaerobic chamber. Bulk solutions were added to the 14 mL conical tubes to provide a final concentration of 100 mM EPPS, 200 mM 10 pyruvate, 100 mM tryptophan, 1 mM MgCl2, 0.05 mM PLP, 0.5 mg / mL D-aminotransferase and 0.01 mg / mL aldolase from SEQ ID NO: 276 or 0.05 mg / mL aldolase HMG from S. meliloti. The proposed enzyme stabilizing components were added in various final concentrations (Tables 38 and 39) to bring the final reaction volume to 7 mL per tube. Reactions were incubated at room temperature in an anaerobic chamber with slight shaking for 24 hours. Samples were extracted periodically and these were analyzed to verify the

Monatin as described in Example 1 using the LC / MS / MS Multiple Reaction Control Procedure. Initial rates were calculated from samples drawn between 0 and 3 hours after addition of the enzyme.

Table 38

Additive

Multiple of the initial monatin formation rate Multiple of monatin final assessment (20 hours)

None

1.0 1.0

Tween 80 0.01% (v / v)

1.3 1.4

Tween 80 0.1% (v / v)

1.3 1.5

Tween 20 0.01% (v / v)

1.1 1.5

Tween X-100 0.01% (v / v)

1.1 1.2

Acetone 5% (v / v)

0.4 0.3

5% ethanol (v / v)

0.7 0.5

Glycerol 1% (w / v)

1.9 1.1

Glycerol 5% (w / v)

1.4 1.4

0 10% glycerol (w / v)

1.1 1.7

Trehalose 10% (w / v)

1.0 1.3

Inositol 10% (w / v)

1.3 1.5

Sorbitol 10% (w / v)

1.1 1.3

Erythritol 10% (w / v)

0.8 1.0

20 Table 39 (SEQ ID NO: 276 aldolase)

Additive

Multiple of the initial monatin formation rate Multiple of the final assessment of monatina at 22 hours

Tween 80 0.01% (v / v)

1.0 1.0

Glycerol 1% (w / v)

1.2 0.9

Glycerol 5% (w / v)

1.5 1.5

Glycerol 10% (w / v)

1.7 2.1

Addition of 0.01% -0.1% (v / v) detergent, such as Triton X-100, Tween 20 or Tween 80, or 1% to 10% (w / v) polyol, such as glycerol, trehalose , inositol or sorbitol, improved the stability of T243N D-aminotransferase for the duration of the experiment.

Example 28 (not claimed)

5 A: Cloning of broad specificity amino acid racemase (BAR) KT2440 from Pseudomonas putida

A BAR was identified in P. putida KT2440 using the information in the literature (Roise, D., Soda, K., Yagi, T., Walsch, CT, Biochemistry 23, 5195-5201, (1984)). . The active site of a P. striata BAR enzyme was sequenced and reported —LTAVLKADAYGXGIGL (SEQ ID NO: 375). This sequence was used for BLAST, the P. putida KT2440 genomic sequence available from NCBI. A protein with a sequence of

10 almost identical consensus. The primers were designed to clone the genomic DNA gene obtained from the American Type Culture Collection (ATCC, Manassas, VA).

(5'-AGAAGACATATGCCCTTTCGCCGTAGGG-3 '(SEQ ID NO: 376 and

5'-AGAAGAGGATCCTCAGTCGACGAGTATCTTCG-3 ') (SEQ ID NO: 377)).

PCR was carried out under standard conditions and the PCR product was purified (PCR purification kit

15 QIAquick, Qiagen, Valencia, CA). The purified PCR product was digested with Nde I and BamH I. The digested PCR product was gel purified (QIAquick gel extraction kit, Qiagen, Valencia, CA) and ligated to pET30 and pET28 that had been digested and gel purified in a similar way. Clones with inserts were sequenced (Agencourt, Beverly, MA) and isolates with the correct sequence (pET30 KT2440 BAR and pET28 KT2440BAR) were identified and used in subsequent studies.

20 The DNA sequence of KT2440 BAR is:

(SEQ ID NO: 378) The amino acid sequence of KT2440 BAR is:

(SEQ ID NO: 379)

B) Purification of KT2440 BAR from P. putida.

The KT2440 BAR pET30 plasmid described above was transformed into DE3 pLysS (Invitrogen, Carlsbad, CA). The resulting strain was grown in LB or Terrific broth at 37 ° C with aeration at OD600nm of 0.4 to 0.6 and induced with 1mM IPTG. Incubation continued for 3 to 4 hours at 37 ° C with aeration. Cells were harvested by centrifugation and the cell pellet was stored at -80 ° C until use. The cell pellet was thawed on ice. Cells were lysed with BugBuster (Novagen, Madison, WI) and benzonase (Novagen, Madison, WI). Cell debris was removed by centrifugation and the cell-free extract was either used immediately or stored at -80 ° C. The KT2440 BAR gene was also cloned into the NdeI-BamHI sites of pET28 and transformed into BL21 DE3 pLysS. This construct did not appear to express soluble proteins very efficiently, so the unlabeled version (pET30 KT2440 BAR) was studied in future studies.

The extract was applied to a UnoQ column (Bio-Rad, Hercules, CA) that had been equilibrated with at least 5 column volumes of buffer A (25 mM potassium phosphate, pH 8.0, 10 µM pyridoxal- 5'-phosphate (PLP)). The column was washed with 2 column volumes of buffer A. The protein was eluted with a linear gradient of buffer B (buffer A + 1M NaCl) from buffer B from 0 to 100% over 20 column volumes and 5ml fractions were collected from the moment the gradient started. Fractions were analyzed using the Amplex Red procedure described in Example 4 Part 7. In summary, 100 µg D-amino acid oxidase (Sigma-Aldrich, St. Louis, MO), 0.05 mM FAD was combined. , 25 mM L-trp and a small volume of the fraction to be analyzed in 50 µL H2O and added to the Amplex Red reaction buffer prepared as indicated in the manufacturer's protocol. The fractions with activity were desalted with a PD-10 column (GE Healthcare, Piscataway, NJ) and concentrated with Amicon centrifuge concentrators (Millipore, Billercia, MA). The purified protein was stored at -80 ° C.

C) Production and assay of Geobacillus stearothermophilus alanine racemase mutant Y354A with tryptophan racemase activity.

Geobacillus stearothermophilus wild-type alanine racemase (SEQ ID NO: 380, shown below, was cloned into pET30 and used as a template for site-directed mutagenesis to effect the Y354A change. The gene of SEQ ID NO: 380 it can be amplified through standard PCR protocols and cloned by using standard recombinant DNA techniques.The gene in SEQ ID NO: 380 can also be reconstructed by any procedure known to those skilled in the art, such as assembly procedures PCR techniques known to those skilled in the art.

The DNA and amino acid sequences of the wild-type alanine racemase from Geobacillus stearothermophilus are shown below as SEQ ID NO: 380:

(SEQ ID NO: 380)

The encoded amino acid sequence of the alanine racemase (Geobacillus stearothermophilus) is shown below as SEQ ID NO: 381:

(SEQ ID NO: 381)

Mutagenesis was performed using the QuickChange multi-site directed mutagenesis kit (Stratagene, La Jolla, CA). The following mutagenic primer was used to make the Y354A change, 5'10 gccatttggaaacgatcaacgcggaagtgccttgcacgatcag-3 '

(SEQ ID NO: 382).

Site-directed mutagenesis was performed as described in the manufacturer's protocol. Several isolates (Agencourt, Beverly, MA) were sequenced and an isolate with the correct sequence was selected and used for further analysis.

5 The single mutant pET30Y354A was transformed into BL21 (DE3) pLysS from E. coli. The purified protein was prepared as follows: The resulting strain was grown in LB or Terrific broth (at 37 ° C with aeration) at OD600nm of 0.4 to 0.6 and induced with 1mM IPTG. Incubation continued at 37 ° C with aeration for approximately 3 hours. Cells were harvested by centrifugation and the cell pellet was stored at -80 ° C.

The cell pellet was thawed on ice and resuspended in a suitable volume of BugBuster

10 (Novagen, Madison, WI) plus Benzonase nuclease (Novagen, Madison, WI). Cell debris was removed by centrifugation and the cell-free extract was applied to an HIS-Bind column (Novagen, Madison, WI) that had been equilibrated with binding buffer. The column was washed with binding buffer and wash buffer and the protein was eluted with elution buffer (as indicated by the manufacturer's protocol). The purified protein was desalted by using a PD-10 column (GE Healthcare, Piscataway, NJ). The protein was desalted in 50 mM phosphate of

15 potassium, pH 8.0 and 10 µM pyridoxal-5'-phosphate according to the manufacturer's protocol. The protein was concentrated using the Amicon centrifuge concentrator (Millipore, Billercia, MA). The purified and concentrated protein was divided into small aliquots and stored at -80 ° C until use.

The purified Y354A is compared to the wild-type alanine racemase (prepared as described above) in the alanine and tryptophan assays. Assays were performed at 37 ° C in 50mM buffer

20 potassium phosphate, pH 8 and 10 µM PLP by using 400 µL of concentrated purified protein (> 1 mg / ml) and 50 mM of substrate. Detection of D-alanine and D-tryptophan was performed using the chiral amino acid methodology described in Example 1.

Table 40

Enzyme

Substratum Weather D isomer produced (ppm)

Wild guy

L-tryptophan 0 na *

10

na

30

na

60

na

1080

na

Y354A

0 na

10

198

30

568

60

1386

1080

10080

Wild guy

L-Alanine 0 5140

10

5960

30

6280

60

6500

1080

5040

Y354A

0 4760

10

4980

Enzyme

Substratum Weather D isomer produced (ppm)

30

4980

60

4200

1080

5000

* na = not detected.

These data were analyzed without the use of an internal standard; therefore these data are semi-quantitative and should be used for comparative purposes. However, these results show that the simple Y354A mutation is sufficient to amplify the specificity of the alanine racemase in such a way that it can catalyze the

5 racemization of amino acids through the use of alternative substrates.

D) BAR enzyme assay.

The Bar enzyme was analyzed in the following way. Amplex Red Assays were prepared as described in this example. KT2440 BAR from P. putida at 200 µg (purified as described in this example) was used. The G. stearothermophilus wild-type alanine racemase and Y354A were purified as described in this example and

10 used at 200 µg / mL or 1000 µg / mL. CE is a cell-free extract that was prepared as described in this example. The results for the 60 minute time interval are shown in Table 41 below.

Table 41

Enzyme

Fluorometer reading (after 60 minutes)

BAR (200)

56943

Y354A (200)

7860

Y354A (1000)

13587

WT alanina racemasa (200)

3646

WT alanina racemasa (1000)

3639

BAR CE (5! L)

16228

BAR CE (10! L)

26662

BAR CE (50! L)

> 58000

Without enzymes

1510

The purified protein was also analyzed to verify the activity of tryptophan racemase in 50 mM potassium phosphate, pH 8, 10 µM PLP and 30 mM L-tryptophan as described above. 200 µg / mL or 1000 µg / mL of purified enzyme was used in the assays (indicated in parentheses). D-Tryptophan was analyzed using the chiral amino acid procedure described in Example 1 for detection.

20 Table 42

Enzyme

Weather D-tryptophan (ppm)

BAR (200)

0 0

5

172

10

410

twenty

844

Enzyme

Weather D-tryptophan (ppm)

30

1318

60

2362

120

2594

240

2762

1080

2294

Y354A (200)

0 0

5

0

10

0

twenty

0

30

12

60

22

120

44

240

56

1080

368

Y354A (1000)

0 0

5

0

10

12

twenty

18

30

40

60

80

120

146

240

218

1080

1164

The assay indicated that the P. putida KT2440 BAR enzyme is much more active on tryptophan than the enzyme derived from G. stearothermophilus and its mutant Y354A.

E) Monatin production with KT2440 BAR from P. putida.

A monatin production assay was performed with the purified P. putida KT2440 BAR (as purified above) (100 µg) or purified Y354A (as purified above) (500 µg), D-aminotransferase (BioCatalytics AT -103 (Pasadena, CA)) (500 µg) and the aldolase of SEQ ID NO: 276) (as purified in Example 23) (50 µg). The monatin production experiment starting with L-tryptophan was performed as follows. In addition to the above enzymes, the following was added per 1 mL of reaction mixture: 4 mM MgCl2,

10 50 mM L-tryptophan, 100 mM sodium pyruvate, 100 mM potassium phosphate buffer, pH 7.5, and 0.05 mM PLP.

As a control, the experiment was performed as described above without racemase and started with D-tryptophan instead of L-tryptophan. Table 43 below summarizes the results.

Table 43

Substratum

 Racemasa Weather Total monatina % R, R % H.H % R, S % MR

L-Trp

Y354A 2 hours None detected

18 hours

None detected

L-Trp

PUB 2 hours None detected

18 hours

38.6 ppm 92.1 5 2.9

L-Trp

None 2 hours None detected

18 hours

None detected

D-trp

None 2 hours 19.9 ppm Without trying Without trying Without trying Without trying

18 hours

221.25 ppm 97.8 0.2 2

Monatin was not detected by using Y354A in this experiment. This racemase was used in the past (data not shown) to produce monatin, but a much higher level of enzyme was used (at least 2 mg and up to 10 mg to see higher monatin levels). The P. putida KT2440 BAR was used to produce monatin from L-tryptophan. The 100 µg used in this experiment was not enough to see monatin production after two hours but it was enough to see monatin production after 18 hours. The distribution of stereoisomers indicated that most of the monatin produced is the R, R isomer. No R, S isomer was produced. This result indicates that KT2440 BAR is unable to detectably racemize the R, R isomer of monatin (racemization of the R, R isomer would produce the R, S isomer). A significant amount of the S, S isomer was produced in this experiment. This is probably due to the fact that the AT103 used in this experiment is not highly purified and may contain L-aminotransferases from the cell extract and there is a large amount of L-tryptophan present to serve as an amino donor for the transamination of S-MP.

Example 29 (not claimed)

Cloning and expression of arginine racemase from Pseudomonas taetrolens

Experimental summary

Pseudomonas taetrolens arginine racemase (also known as P. graveolens) (Genbank Accession No. AB096176, nucleic acid sequence) and its 1384M mutant were cloned, expressed and analyzed to verify their activity in L-tryptophan conversion D-tryptophan. This gene is 72% identical to the BAR gene of

P. putida from KT2440 and 73% identical to the BAR gene from P. putida from NBRC 12996 described above. The amino acid sequence is 72% identical to both P. putida BAR proteins.

Polymerase chain reaction protocol

Pseudomonas taetrolens (ATCC 4683) was grown in nutrient broth at 28 ° C with shaking at 225 rpm. Polymerase chain reaction was performed on all cells using primers designed with 5 'restriction sites and overhangs to clone vectors pET 28 and pET30 (Novagen, Madison, WI).

The primer sequences were as follows:

Terminal N: 5'-ATAATACATATGCCCTTCTCCCGTACCC-3 '(SEQ ID NO: 408) and

Terminal C: 5'-GCGGCGGGATCCTTACTGATCTTTCAGGATT-3 '(SEQ ID NO: 409).

The P. taetrolens derived gene was amplified using the following PCR protocol. Twenty-five µL of cultured cells were lysed at 96 ° C for 10 minutes. Cell debris was removed by centrifugation and the supernatant was used as a template for the PCR. The 100 µL reaction contained 5 µL of template (lysed cell supernatant), 1.6 µM of each primer, 0.3 µM of each dNTP, 10 U of rTth Polymerase XL (Applied Biosystems, Foster City, CA ), 1 × XL buffer and 1 mM Mg (OAc) 2. The thermal cycler program used included a warm start at 94ºC for 3 minutes, 8 repetitions of the following stages: 94ºC for 30 seconds, 52ºC for 30 seconds and 68ºC for 2 minutes, followed by 22 repetitions of the following stages: 94ºC for 30 seconds , 58ºC for 30 seconds and 68ºC for 2 minutes. After repetitions the sample was kept at 68 ° C for 7 minutes and then stored at 4 ° C. This PCR protocol produced a 1230 bp product.

Cloning

PCR products were purified on 0.8% TAE-agarose gels using the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA). The product was cloned into TOPO and transformed into TOP 10 cells according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from the resulting transformants using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with Nde I and BamH I. Plasmid sequences that they appear to have a correct insertion were verified by dideoxy chain termination DNA sequencing with the forward M13 and reverse M13 universal primers.

The correct TOPO clone was digested with restriction enzymes Nde I and BamH I according to the manufacturer's recommended protocols (Biolaboratories of New England, Beverly, MA) and purified on 0.8% TAEagarose gels using the kit. gel extraction from Qiagen (Qiagen, Valencia, CA). Vectors pET28 and pET30 were prepared by restriction enzyme Nde I and BamH I digestion followed by treatment with serum alkaline phosphatase (Roche, Indianapolis, IN) and purification on 0.8% TAE-agarose gels using the Qiagen gel extraction kit (Qiagen, Valencia, CA). The digested vectors and inserts were ligated using the DNA Rapid Ligation Kit (Roche, Indianapolis, IN). Approximately 50 ng of the treated insert, 100 ng of the treated vector (3 to 1 insert to vector ratio), 5 U of T4 DNA ligase and 1 × ligation buffer were incubated for 5 minutes at room temperature. The ligation reaction was desalted using the highly pure PCR product purification kit (Roche, Indianapolis, IN) and used to transform the E. coli DH10B electrocompetent cells (Invitrogen, Carlsbad, CA). 10 µl of each ligation reaction was added to 40 µl of DH10B cells and transformed by electroporation using the Bio-Rad II gene driver under the following conditions: 2.5 kV, 25 ° F, 200 ohm in a 0.2 cm cuvette. The cells were allowed to recover in 1 mL of SOC medium at room temperature for 1 hour at 37 ° C with stirring at 225 rpm. Cells were plated on LB plates containing kanamycin (50 µg / mL). Plasmid DNA was purified from the resulting transformants using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with Nde I and BamH I.

Gene expression and analysis

Plasmid DNA was transformed into the E. coli BL21 (DE3) pLysS expression host (Novagen, Madison, WI). Cultures were grown and plasmids were isolated using the Qiagen miniprep kit (Qiagen, Valencia, CA) and analyzed by restriction enzymes to confirm identity.

Induction into BL21DE3 pLysS was initially performed in both vectors pET 28 (histidine labeled) and pET 30 (unlabeled). A temporary study was performed with cultures grown at 37ºC in 100 mL of kanamycin containing LB (50 mg / L) at an OD600 of 0.5, induced with 100 mM IPTG (isopropyl thiogalactoside) and sampled at 0 and 3 hours after induction. Cells from hour 0 and hour 3 were resuspended in 1 × sodium dodecyl sulfate buffer containing 2-mercaptoethanol and heated at 95 ° C for 10 minutes and cooled. Aliquots of these whole cell protein samples were analyzed by SDS-PAGE using a 4 to 15% gradient gel.

Cell extracts from the 3-hour cultures were also prepared by suspending cell pellets from 5 ml of culture in Novagen's BugBusterTM reagent containing benzonase nuclease and group # 3 protease inhibitor mixture (Calbiochem-Novabiochem Corp ., San Diego, CA) at room temperature for 20 minutes with light agitation and centrifugation at 16,000 xg to remove cell debris. Supernatants (cell extracts) were loaded onto 4 to 15% gradient gels for analysis of soluble cell proteins.

The 3 hour sample of the cloned P. taetrolens arginine racemase showed a total protein band that corresponded to the correct size (approximately 45 kDa) in vector pET 30 (unlabeled). The P. taetrolens gene product in pET 30 was overexpressed to a higher level than the P. taetrolens gene product in pET 28 (histidine labeled), but none of the vectors provided a visible soluble protein band.

Cells from the induced cultures (100 mL) were centrifuged and washed once with 0.85% NaCl. The cell pellets were resuspended in 5 mL / g wet cells of BugBusterTM Reagent (Novagen, Madison, WI) containing 5 µl / ml of set # 3 protease inhibitor mixture (Calbiochem-Novabiochem Corp ., San Diego, CA) and 1 µl / ml of benzase nuclease. The samples were incubated at temperature

room for 20 minutes on an orbital shaker. Insoluble cell debris was removed by centrifugation at

16,000 x g for 20 minutes at 4 ° C.

Cell extracts were analyzed to verify tryptophan racemase activity using the following protocol. Reactions of 1 mL were carried out in 50 mM of potassium phosphate (pH 8.0), 0.05 mM of 5 PLP and 30 mM L-tryptophan. Reactions were started with the addition of cell-free extracts and incubated at 30 ° C overnight. Aliquots of the samples were taken after incubation overnight (the zero minute samples served as a control reaction). Concentrated formic acid (5 µL) was added to each 250 µL sample aliquot to stop the reaction and the precipitated protein was removed by centrifugation. Supernatants were removed and frozen at −80 ° C until analyzed for D-tryptophan by

10 the chiral amino acid procedure described in Example 1.

The results of assays of the cell extracts of depET28 and pET30 induction with 100 mM IPTG (3 hours) demonstrated that the P. taetrolens clones show racemase activity in L-tryptophan. Again, the tagged version of BAR appears to be less active and may precipitate or be less soluble than the unchecked version (pET28). Table 44 below shows the initial results, although not quantitative since they are

15 obtained a very poorly soluble protein.

Table 44

Treatment

Time interval Substratum Racemase extract (200! G) D-trp conc (! G / mL)

pET28 / P. taetrolens

0 L-trp 500! L na

pET30 / P. taetrolens

0 L-trp 500! L na

pET28 / P. taetrolens

overnight L-trp 500! L 140

pET30 / P. taetrolens

overnight L-trp 500! L 226

The induction of the construction of pET30 (unlabelled) was repeated using the same conditions as mentioned above and a band of soluble proteins visible on SDS-PAGE was observed. The test was repeated using the same conditions as described above and the results were obtained as shown in Table 45 below.

Table 45

Treatment

Time interval Substratum Racemase extract (! L) D-trp conc (! G / mL)

P. taetrolenspET30

0 L-trp 300 Nd

P. taetrolenspET30

0 L-trp 150 Nd

P. taetrolenspET30

2 hours L-trp 300 319

P. taetrolenspET30

2 hours L-trp 150 308

P. taetrolenspET30

Overnight L-trp 300 1586

P. taetrolenspET30

Overnight L-trp 150 1658

Again, it was observed that doubling of volumes does not escalate to increased activity. For future work, the extraction of the Bugbuster protein was determined as quickly as possible after the preparation of the cell extracts and to store the protein in 50 mM phosphate buffer, pH 8 containing 0.01 mM PLP. The detergent in Bugbuster may inhibit the reaction or may cause loss of activity after

5 storage.

The induction of the pET30 construct was carried out again and the cell extract was processed with anion exchange chromatography (as in Example 28) to provide a pure extract. The test was repeated with this partially purified preparation. The numbers in the parentheses in the racemase extract column in Table 46 below indicate the approximate amount of racemase enzyme partially

Purified 10 used in the assay. The test results are shown in Table 46 below.

Table 46

Enzyme source

Time interval Substratum Racemase extract D-trp conc (! G / mL)

KT2440

0 L-trp 75! L (90! G) na

NBRC 12996

0 L-trp 42! L (200! G) na

NBRC 12996

0 L-trp 21! L (100! G) na

P. taetrolens

0 L-trp 108! L (200! G) na

P. taetrolens

0 L-trp 54! L (100! G) na

KT2440

2 hours L-trp 75! L (90! G) 661

NBRC 12996

2 hours L-trp 42! L (200! G) 408

NBRC 12996

2 hours L-trp 21! L (100! G) 208

P. taetrolens

2 hours L-trp 108! L (200! G) 862

P. taetrolens

2 hours L-trp 54! L (100! G) 547

KT2440

overnight L-trp 75! L (90! G) 2386

NBRC 12996

overnight L-trp 42! L (200! G) 2382

NBRC 12996

overnight L-trp 21! L (100! G) 1706

P. taetrolens

overnight L-trp 108! L (200! G) 2029

P. taetrolens

overnight L-trp 54! L (100! G) 2099

The nonlinearity of the overnight sample in this case is probably due to the fact that the

15 reactions are reaching balance. Clearly, P. taetrolens BAR has significant activity for tryptophan racemization, as do 12996 BAR and KT2440 BAR. It seems that the KT2440 BAR and the BAR of

P. taetrolens have a similar activity, which is slightly higher than that of 12996 BAR.

The DNA sequence of the P. taetrolens arginine racemase is shown below as SEQ ID NO: 410. The PCR sequence provided two changes compared to the published NCBI sequence.

20 Specifically, the PCR sequence contained an adenosine instead of a guanine at position 902 and a cytosine instead of a guanine at position 921. These DNA changes resulted in a silent mutation as well as a change from glycine to aspartate at amino acid position 301.

(SEQ ID NO: 410)

The protein encoded by the gene in SEQ ID NO: 410 was analyzed by the Signal P 3.0 signal peptide prediction program (www.cbs.dtu.dk/services/SignalP/) and a leader sequence of 23 amino acids was predicted. I384M P. taetrolens BAR Mutagenesis Mutagenesis was performed using the QuickChange multi-site directed mutagenesis kit.

(Stratagene, La Jolla, CA), by using the P. taetrolens BAR gene in pET30 resulting in a protein

unmarked. The following mutagenic primer was used to make the 1384M change: 5'-TACCCAGGCTGAGATGGAAGACATCAACG-3 '(SEQ ID NO: 411).

Site-directed mutagenesis was performed as described in the manufacturer's protocol. Several isolates (Agencourt, Beverly, MA) were sequenced and an isolate with the correct sequence was selected and used for further analysis.

The plasmid was transformed into BL21 (DE3) cells (Novagen, Madison, WI). The recombinant protein was produced in Overnight Express II medium (Novagen, Madison, WI) containing 50 µg / mL kanamycin according to the manufacturer's protocols. Cell-free extracts were prepared using BugBuster (Novagen, Madison, WI) according to the manufacturer's protocols, desalted, and analyzed to verify the percent expression of the target protein using the Experion procedure described above. .

All protein assays were performed using the Pierce BCA Kit (Pierce, Rockford, IL). Tryptophan racemase assays were performed with the mutant enzyme using the wild-type enzyme prepared in the same manner as a positive control. The assays contained per mL: 30 mM L-tryptophan, 50 mM potassium phosphate, pH 8, 10 µM PLP, and approximately 100 µg of racemase protein in a cell-free extract. In the case where 100 µg was not used (based on the percentage of expression of Experion and the total protein numbers of Pierce), the results were normalized. Samples were taken at 0 minute, 30 minute, 2 hour, and overnight, treated with 2% formic acid, filtered, and diluted 1:10 for analysis using the chiral amino acid procedure described in example 1.

The wild-type enzyme appeared to produce 49.1 ppm D-tryptophan in 30 minutes, while the 1384M mutant produced 108 ppm. The 2 hour time interval was similar, the wild-type enzyme produced 229.4 ppm Dtryptophan versus 541.7 for the 1384M mutant. The 1384M mutation appears to have approximately doubled the BAR activity of P. taetrolens. The time interval during the night for the mutant is also longer, but as the reactions approach equilibrium the difference between activities decreases. When assayed for monatin production as in Example 28, 1384M did not appear to provide any benefit over P. taetrolens wild-type enzyme.

Example 30 (not claimed)

A. caviae extract assay

Aeromonas caviae ATCC 14486 was grown in a nutrient broth at 37 ° C. The cells of the culture (200 mL) were centrifuged and washed once with 0.85% NaCl. The cell pellets were resuspended in 5 mL / g wet cells of BugBusterTM Reagent (Novagen, Madison, WI) containing 5 µl / ml of set # 3 protease inhibitor mixture (Calbiochem-Novabiochem Corp ., San Diego, CA) and 1 µl / ml of benzase nuclease. The samples were incubated at room temperature for 20 minutes on an orbital shaker. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 min at 4 ° C. The cell free extract was desalted on a PD-10 column (GE Healthcare, Piscataway, NJ).

Cell-free extracts were analyzed to verify tryptophan racemase activity using the following protocol. Reactions of 1 mL were carried out in 50 mM potassium phosphate (pH 8.0), 0.05 mM PLP and 30 mM L-tryptophan. Reactions were initiated with the addition of cell free extracts (100 µL or 500 µL) and incubated at 30 ° C overnight. Aliquots of the samples were taken at 2 hours and after incubation overnight (the zero minute samples served as control reactions). Concentrated formic acid (5 µL) was added to each 250 µL sample aliquot to stop the reaction and the precipitated protein was removed by centrifugation. Supernatants were removed and frozen at −80 ° C until analyzed for D-tryptophan by the chiral amino acid procedure described in Example 1.

Results of the assay of A. caviae cell extracts demonstrated racemase activity on L-tryptophan as shown in Table 47.

Table 47

Treatment

Time interval substratum Racemase extract D-trp conc (! G / mL)

A. caviae

0 L-trp 100! L na

A. caviae

0 L-trp 500! L na

A. caviae

2 hours L-trp 100! L 2

A. caviae

2 hours L-trp 500! L 19

Treatment

Time interval substratum Racemasa extract of D-trp conc (! G / mL)

A. caviae

overnight L-trp 100! L Four. Five

A. caviae

overnight L-trp 500! L 130

After finding activity in A. caviae cell extracts, degenerate primers (based on conserved regions of known BAR homologs) were designed to obtain the BAR gene from these species. The degenerate primer sequences are shown below:

Aer deg F2: 5'-GCCAGCAACGARGARGCMCGCGT-3 '(SEQ ID NO: 412); and

Aer deg R1: 5'-TGGCCSTKGATCAGCACA-3 '(SEQ ID NO: 413)

where K indicates G or T, R indicates A or G, S indicates C or G, and M indicates A or C.

The above primers were used to amplify the PCR to 715 bp DNA fragments of A. caviae genomic DNA (ATCC 14486). The following PCR protocol was used: The 50 µL reaction contained 0.5 10 µL of template (approximately 100 ng of A. caviae genomic DNA), 1.6 µM of each primer, 0.3 mM of each dNTP, 10 U of rTth Polymerase XL (Applied Biosystems, Foster City, CA), 1 × XL buffer, 1 mM Mg (OAc) 2 2.5 µL dimethyl sulfoxide. The thermocycler program used included a hot start at 94 ° C for 3 minutes and 30 repetitions of the following steps: 94 ° C for 30 seconds, 53 ° C for 30 seconds and 68 ° C for 2 minutes. After 30 repetitions the sample was kept at 68ºC for 7 minutes and at

It was then stored at 4 ° C. This PCR protocol generated the 715 bp product.

Cloning

PCR products were purified on 0.8% TAE-agarose gels using the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA). The product was cloned into TOPO and transformed into TOP 10 cells according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from transformants

20 resulting using the Qiagen spin miniprep kit (Qiagen, Valencia, CA) and analyzed for correct insertions by restriction enzymes with EcoR 1. Plasmid sequences that appear to have a correct insert were verified by sequencing of Dideoxy chain termination DNA with direct M13 universal primers.

The DNA sequence of the A. caviae PCR product is shown below as SEQ ID NO: 414), 25 where the degenerate primer sequence regions are underlined:

(SEQ ID NO: 414), where R indicates A or G, S indicates C or G, and M indicates A or C.

The amino acid sequence of the partial enzyme BAR of A. caviae is shown as SEQ ID NO: 415 below.

where X is H, Q, N or K (SEQ ID NO: 415).

The consensus protein fragment of SEQ ID NO: 415 is more than 89% homologous at the amino acid level with respect to the published TIGR sequence for A. hydrophila. Because the highly related Aeromonas hydrophila protein exhibited broadly specific racemase activity, as well as the 10 A. caviae cell extracts, it was expected that the full length coding region for A. caviae, once obtained, would produce a racemase that would also have broad specificity with activity on tryptophan. Genomic walk procedures were used to obtain the full-length gene sequence of the BAR gene from

A. caviae shown below as SEQ ID NO: 416.

(SEQ ID NO: 416). The amino acid sequence for the native BAR of A. caviae is shown below as SEQ ID NO: 417.

5 1 mhkktllatl ifgllagqav aapylpladd hrngqeqtaa 41 nawlevdlga fehniqtlkn rlgdkgpqic aimkadaygh 81 gidllvpsvv kagipcigia sneearvare kgfegrlmrv 121 raatpdeveq alpykleeli gslesakgia diaqrhhtni 161 pvhiglnsag msrngidlrq ddakadalam lklkgitpvg

10 201 imthfpveek edvklglaqf kldyqwlida gkldrsklti 241 haansfatle vpeayfdmvr pggiiygdti psyteykkvm 281 afktqvasvn hypagvvkkvkgkkvkgvkkvkgkvkgvvkgvqkvvgkvkvgvkvgvkvgvkvvvhvqvqvv

15 401 tnpkkikr. (SEQ ID NO: 417)

The first 21 N-terminal amino acid residues of SEQ ID NO: 417 are predicted to be a signal peptide using the Signal P 3.0 program (www.cbs.dtu.dk/services/SignalP/). Experimental tests confirmed that the expression product was secreted into the E. coli periplasm and the signal peptide was cleaved as predicted. When the full length gene was cloned and expressed using the procedures described above, it was found to have comparable activity, but greater than that of the P. taetrolens BAR.

Example 31

Production of the aldolase from SEQ ID NO: 276 in an alternative expression host

The gene in SEQ ID NO: 275 was subcloned using standard molecular biology procedures into a derivative of the pET23d vector (Novagen, Madison, WI) containing the E. coli metE gene and the promoter inserted at the site of NgoMIV restriction and a second pis restriction site that was added for easy extraction of the beta lactamase gene (baa). The construction of this vector containing an insert for a myoinositol oxygenase gene is described in PCT WO2006066072 in Examples 2 and 20. The aldolase insert was confirmed by DNA sequencing (Agencourt Bioscience Corporation; Beverly, MA) and plasmid with the correct insertion sequence it was transformed into the E. coli expression receptor BW30384 (DE3) �ompT�metE. The construction of this expression host and the transformation protocol are also described in PCT WO2006066072 (examples 21 and 22). The aldolase gene was expressed using the Novagen Overnight Express ™ II autoinduction system (Novagen, Madison, WI) containing 100 mg / L ampicillin. This system was described in Example 24 for the expression of Bacillus sphaericus D-alanine aminotransferase (strain ATCC 10208). Cell-free extracts containing aldolase were produced using Novagen's BugBuster ™ Extraction Reagent (Primary Amine Free) (Novagen, Madison, WI) containing 1 µL / mL benzase nuclease, 0.033 µL / mL of r-Lysozyme and 5 µL / mL of set II protease inhibitor mixture according to the manufacturer's recommended protocol for cell lysis. The soluble proteins in the cell-free extracts were separated on a Bio-Rad Experion Automated Electrophoresis Station (Bio-Rad, Hercules, CA) and analyzed to verify the percentage of expression of soluble proteins using the Experion version software. 1.1.98.0 as described in Example 12.

To try to improve aldolase expression in E. coli, codons two to seven of the DNA coding sequence were mutated for the suggested changes in wild type sequence analysis using the E. coli ProteoExpert RTS HY algorithm Roche. These changes were made using either the QuikChange® Multiple Site-Directed Mutagenesis Kit or the QuikChange® II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's suggested protocol at 0.75! L of Quik solution every 25 µL of reaction mix and transformation to ultracompetent XL10-Gold® cells. Mutations were generated using primers (every 45 nucleotides in length) designed with Stratagene's QuikChange® primer design website program available online at www.stratagene.com.qcprimerdesign.

Table 48

Codon 1

Codon 2 Codon 3 Codon 4 Codon 5 Codon 6 Codon 7

Wild guy

ATG CCT ATC GTT GTT ACG AAG

ProteoExpert # 1

ATG CCA ATT GTT Gta ACT AAA

ProteoExpert # 2

ATG CCA ATT GTT GTT ACT AAA

ProteoExpert # 6

ATG CCA ATT GTT Gta ACC AAA

ProteoExpert # 10

ATG CCA ATT GTT GTT ACC AAA

The aldolase gene sequences with the above codon changes were transformed into the E. coli expression host BW30384 (DE3) �ompT�metE and then expressed using the Novagen Overnight Express ™ autoinduction system 2 (Novagen, Madison, WI). None of these mutations resulted in higher levels of gene expression when compared to the wild type sequence. The typical results of the cell-free extracts were analyzed using Bio-Rad's 1.1.98.0 Experion Pro260 software shown in Table 49 below, where the column titled protein expression shows the values for the total% of soluble proteins.

Cell-free extracts (0.025 mg / mL soluble protein per assay) were analyzed to verify their ability to produce (R, R) -monatin from 200 mM sodium pyruvate and 100 mM D-tryptophan using of the protocol described in Example 7. Reactions (7 mL total volume) were carried out in 14 mL polypropylene tubes in an anaerobic chamber using purified B. sphaericus D-aminotransferase (ATCC 10208) at a final concentration of 2 mg / mL. The monatin concentration produced at 1, 4, and 20 hours after addition of the enzymes is shown in Table 49 below. Table 49 shows that the cell-free extract generated from the construct containing the wild-type aldolase sequence produced a slightly higher 20-hour monatin concentration than the cell-free extracts from the constructs that they had the ProteoExpert mutations. Monatin concentrations were determined by the LC / MS / MS procedure described in Example 1.

Table 49

 Protein expression (%)

[Monatina] mM-1 hour [Monatina]; mM-4 hours [Monatina] mM-20 hours

Wild guy

22 2.1 4.2 13.4

ProteoExpert # 1

18 1.3 3.1 12.4

ProteoExpert # 2

18 2.1 4.7 11.6

ProteoExpert # 6

18 1.4 1.9 12.4

ProteoExpert # 10

twenty 1.8 1.8 11.7

These data demonstrate that the aldolase of SEQ ID NO: 276 can be produced in an alternate expression host without IPTG induction.

The bla gene was extracted from the vector and subsequent fermentations to produce aldolase were performed without the use of an antibiotic as described in PCT WO2006066072 for another enzyme. Expression levels in the fermentations per batch fed at 30 ° C peaked at 6 to 8 hours after induction, resulting in the aldolase of SEQ ID NO: 276 at 25-30% soluble protein, according to Experion data. Stability studies showed no apparent loss of aldolase activity when the fermentation product was left for 6 hours at 15 or 30 ° C (under oxygen limited conditions as well as under aerated conditions) before cell concentration and disruption. Aldolase was found to be equally stable when stored as a cell-free extract or as a cell pellet when stored for 5 days at −80 ° C. Washing the cell pellet prior to storage at −80 ° C was not required and actually caused a small decrease in activity. Cells resuspended in distilled water, fermentation supernatant, or 100 mM potassium phosphate buffer (pH 7.8) were found to show no loss in activity or protein concentration (judged by SDS- PAGE) when stored for 11 days at room temperature or at 4 ° C. The cell-free extract produced in potassium phosphate buffer showed no loss of aldolase activity when stored at 4 ° C or at room temperature for 5 days. Cells can be opened in phosphate buffer, up to 25% supernatant or water with comparable recovery of aldolase activity; however, the addition of 1mM MgCl2 to water was found to slightly improve aldolase activity. These data show that the protein aldolase is stable enough to be commercially useful.

SEQUENCE LIST

<110> Burke, Ellen Gort, Steven John Hicks, Paula M. Luginbuhl, Peter McFarlan, Sara C. Richardson, Toby Solheid, Christopher Weiner, David Zhao, Lishan

<120> Aldolases, nucleic acids encoding them, and methods of producing and using them

10

<130> 2464.0190002 / MAC / M-R <140> Will be assigned <141> Attachment

fifteen

<150> 60 / 853,005 <151>

<150> 60 / 799,460 <151>

twenty

<160> 417

<170> PatentIn 3.1

25

<210> 1 <211> 699 <212> DNA <213> Bacteria

30

<400> 1

35

<210> 2 <211> 232 <212> PRT <213> Unknown

40

<220> 201

<223> Isolated

<220> 5 <221> DOMAIN

<222> (8) ... (165)

<223> Desmethylmenaquinone methyltransferase

10 <400> 2 5 <210> 3

<211> 693

<212> DNA

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<400> 3

15 20

<210> 4 <211> 230 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

25

<220> <221> DOMAIN <222> (28) ... (174) <223> Desmethylmenaquinone methyltransferase

<400> 4

<210> 5

<211> 762

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 6

<211> 253

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (191)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 6

<210> 7

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 7 5 <210> 8

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 8 <210> 9

<211> 552

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 9 <210> 11

<210> 10

<211> 184

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (173)

twenty

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (2) ... (5)

25

<223> N-glycosylation site. Prosite id = PS00001

<400> 10

<211> 645

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 11

<210> 12

<211> 214

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (10) ... (162)

<223> Desmethylmenaquinone methyltransferase

20 <400> 12

<210> 13

<211> 498 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 13

<210> 14 15 <211> 165

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<400> 14

5

<210> 15

<211> 723

<212> DNA

<213> Unknown

10

<220>

<223> Obtained from an environmental sample

<400> 15

5 <210> 16

<211> 240

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (19) ... (188)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 20 <222> (155) ... (158)

<223> N-glycosylation site. Prosite id = PS00001

<400> 16

<210> 17

<211> 792 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 17 5 <210> 18

<211> 263

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (39) ... (205)

<223> Desmethylmenaquinone methyltransferase

<400> 18

<210> 19

<211> 723 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 19 <210> 20

<211> 240

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

10 <221> DOMAIN

<222> (30) ... (197)

<223> Desmethylmenaquinone methyltransferase

<220>

15 <221> SITE

<222> (221) ... (224)

<223> N-glycosylation site. Prosite id = PS00001

<400> 20

<210> 21

<211> 444 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 21

<210> 22 15 <211> 148

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<400> 22 5 <210> 23

<211> 801

<212> DNA

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<400> 23

<210> 24

<211> 266

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (36) ... (207)

fifteen

<223> Desmethylmenaquinone methyltransferase

<400> 24

<210> 25

<211> 741 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 25

<210> 26 15 <211> 246

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (38) ... (202) 25 <223> Desmethylmenaquinone methyltransferase <220>

<221> SITE

<222> (119) ... (122)

<223> N-glycosylation site. Prosite id = PS00001

<220>

<221> SITE

<222> (179) ... (182)

<223> N-glycosylation site. Prosite id = PS00001

<400> 26

<210> 27

<211> 693 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 27

<210> 28 15 <211> 230

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (27) ... (179) 25 <223> Desmethylmenaquinone methyltransferase

<400> 28 <210> 29

<211> 756

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 29

<210> 30

<211> 251

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (28) ... (184)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 30

<210> 31

<211>

 327 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 31

<210> 32

<211>

 109 5 <212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 32

<210> 33 15 <211> 681

<212> DNA

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<400> 33 5 <210> 34

<211> 226

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (3) ... (171)

<223> Desmethylmenaquinone methyltransferase

<400> 34

<210> 35

<211> 681 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 35

<210> 36 15 <211> 226

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 36 <210> 37

<211> 675

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 37

<210> 38 10 <211> 224

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (173) 20 <223> Desmethylmenaquinone methyltransferase

<400> 38 <210> 39

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 39 5 <210> 40

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 40

<210> 41

<211> 672

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 43

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 43

<210> 45

<211> 672

<212>

 DNA 5 <213> Unknown

10 <400> 41

<210> 42

<211> 223

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

25

<223> Desmethylmenaquinone methyltransferase

<400> 42

10 15

<210> 44 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

<400> 44

<220>

<223> Obtained from an environmental sample

10 <400> 45

15 <210> 46

<211> 223

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 46

<210> 47

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 47

<210> 48 15 <211> 223

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 25 <223> Desmethylmenaquinone methyltransferase

<400> 48 <210> 49

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 49

10 <210> 50

<211> 223

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 50

<210> 51

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 51

<210> 52

<211> 223

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

fifteen

<223> Desmethylmenaquinone methyltransferase

<400> 52

<210> 53

<211> 675

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 53

<210> 54

<211> 224

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (21) ... (173)

<223> Desmethylmenaquinone methyltransferase

25 <400> 54 <210> 55

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 55 5 <210> 56

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 56

<210> 57

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 57 <210> 58

<211> 225

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220> 10 <221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 58 <210> 59

<211> 678

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 59 <210> 61

<210> 60

<211> 225

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 60

<211> 675 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 61 5 <210> 62

<211> 224

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (21) ... (173)

<223> Desmethylmenaquinone methyltransferase

<400> 62

5

<210> 63 <211> 693 <212> DNA <213> Unknown

10

<220>

270

<223> Obtained from an environmental sample

<400> 63 <210> 65

5 10

<210> 64 <211> 230 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

fifteen

<220> <221> DOMAIN <222> (21) ... (173) <223> Desmethylmenaquinone methyltransferase

<400> 64

<211> 672

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400>

 65

<400>

 67

10

<210> 66 <211> 223 <212> PRT <213> Unknown

fifteen

<220> <223> Obtained from an environmental sample

20 25

<220> <221> SIGNAL <222> (1) ... (27) <220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase <400> 66

5

<210> 67 <211> 672 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample

274

5 10

<210> 68 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

fifteen

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

<400> 68

<210> 69 <211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 69

10 15

<210> 70 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

25

<220> <221> SITE <222> (149) ... (152) <223> N-glycosylation site. Prosite id = PS00001

<400> 70

5

<210> 71 <211> 672 <212> DNA <213> Unknown

10

<220>

278

<223> Obtained from an environmental sample

<400> 71 <210> 73

5 10

<210> 72 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

fifteen

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

<400> 72

<211> 690

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 73

10 <210> 74

<211> 229

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 74

<210> 75

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 75 5 <210> 76

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 76 <210> 77

<211> 681 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 77

10 <210> 78

<211> 226

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 78 <210> 79

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 79

5 10

<210> 80 <211> 225 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

fifteen

<220> <221> DOMAIN <222> (22) ... (174) <223> Desmethylmenaquinone methyltransferase

<400> 80

<210> 81

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 81

15 <210> 82

<211> 225

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 82

<210> 83

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 83

<210> 84 15 <211> 225

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 25 <223> Desmethylmenaquinone methyltransferase

<400> 84

<210> 85

<211> 699

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 87

<211> 672

<212>

 DNA 5 <213> Unknown

10 <400> 85

<210> 86

<211> 232

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (173)

25

<223> Desmethylmenaquinone methyltransferase

<400> 86

<220>

<223> Obtained from an environmental sample

10 <400> 87

15 <210> 88

<211> 223

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 88 <210> 89

<211> 696 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 89

10 <210> 90

<211> 231

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 90

<210> 91

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 91

<210> 92

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 92

<210> 93

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 93 5 <210> 94

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 94

<210> 95

<211> 639 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 95

<210> 96 15 <211> 212

<212> PRT

<213> Unknown <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (9) ... (161)

<223> Desmethylmenaquinone methyltransferase

<400> 96

<210> 97

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 97

<210> 98

<211> 225

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

25 <400> 98

<210> 99

<211> 639 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 99 5 <210> 100

<211> 212

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (9) ... (161)

<223> Desmethylmenaquinone methyltransferase

<400> 100 <210> 101

<211> 678

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 102

<211> 225

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (28) ... (174)

<223> Desmethylmenaquinone methyltransferase

20 <400> 102

<210> 103

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 103

<210> 104

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 104

<210> 105

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 105 5 <210> 106

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 106

<210> 107

<211> 636 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 107

<210>

 108 15 <211> 211

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (9) ... (161)

<223> Desmethylmenaquinone methyltransferase

<400> 108

<210> 109

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 109

<210>

 110 10 <211> 225

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 20 <223> Desmethylmenaquinone methyltransferase

<400> 110

<210> 111

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 111 5 <210> 112

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 112

<210> 113

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 113

<210> 114

<211> 225

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

25 <400> 114

Leu 225

<210> 115

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 115

10 <210> 116

<211> 223

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 116 <210> 117

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 117 5 <210> 118

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 118 <210> 119 <211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 119 <210> 121

10 15

<210> 120 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

<400> 120

<211> 696 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 121

10 <210> 122

<211> 231

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 122

<210> 123

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 123 5 <210> 124

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 124

<210> 125

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 125

<210> 126

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 126

To be 225

<210> 127 5 <211> 678

<212> DNA

<213> Unknown

<220> 10 <223> Obtained from an environmental sample

<400> 127

15 <210> 128

<211> 225

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 128

5

<210> 129 <211> 678 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample <400> 129

344

5 <210> 130

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 130 <210> 131

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 131

<210> 132 10 <211> 223

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 20 <223> Desmethylmenaquinone methyltransferase

<400> 132

5

<210> 133 <211> 672 <212> DNA <213> Unknown

10

<220>

348

<223> Obtained from an environmental sample

<400> 133 <210> 135

5 10

<210> 134 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

fifteen

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

twenty

<220> <221> SITE <222> (193) ... (196) <223> N-glycosylation site. Prosite id = PS00001

25

<220> <221> SITE <222> (204) ... (207) <223> N-glycosylation site. Prosite id = PS00001

<400> 134

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 136

<211> 223

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

20 <400> 136

<210> 137

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 137

<210> 138 15 <211> 223

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222>

 (20) ... (172) 25 <223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222>

 (149) ... (152) 30 <223> N-glycosylation site. Prosite id = PS00001

<400> 138

<210> 139

<211> 693

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 139

<210> 140

<211> 230

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (27) ... (179)

<223> Desmethylmenaquinone methyltransferase

25 <400> 140

<210> 141

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 141

<210> 142

<211> 223

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

25 <400> 142

5

<210> 143 <211> 672 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample 358

<400> 143

<210> 144

<211> 223

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (1) ... (21)

<223> Reason for signature 1 of the TonB-dependent receptor proteins. Prosite id = PS00430

<400> 144 <210> 145

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 145 <210> 147

<210> 146

<211> 223

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 146

<211> 675

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 147

<210> 148 10 <211> 224

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (173) 20 <223> Desmethylmenaquinone methyltransferase

<400> 148 <210> 149

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 149 5 <210> 150

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 150

<210> 151

<211> 675

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 151

<210> 152

<211> 224

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (21) ... (173)

<223> Desmethylmenaquinone methyltransferase

25 <400> 152 <210> 153

<211> 672

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 153

<210> 154 10 <211> 223

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 20 <223> Desmethylmenaquinone methyltransferase

<400> 154 <210> 155

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 155

10 <210> 156

<211> 223

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 20 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 25 <222> (204) ... (207)

<223> N-glycosylation site. Prosite id = PS00001

<400> 156 <210> 157

<211> 690

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 158

<211> 229

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

20 <220>

<221> SITE

<222> (20) ... (40)

<223> Reason for signature 1 of multi-copper oxidases. Prosite id = PS00079

25 <400> 158 <210> 159

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 159 <210> 161

<210> 160

<211> 223

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 160

<211>

 699 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 161

<210> 163

<211>

 699 5 <212> DNA

<210> 162

<211> 232

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

fifteen

<223> Desmethylmenaquinone methyltransferase

<400> 162

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 163

<210> 164 15 <211> 232

<212> PRT

<213> Unknown

5 10

<220> <223> Obtained from an environmental sample <220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase <400> 164

<210> 165

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 165

<210> 166 15 <211> 223

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 25 <223> Desmethylmenaquinone methyltransferase

<400> 166 <210> 167

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 167 5 <210> 168

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 168

<210> 169

<211> 672 2

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 169 <210> 171

<210> 170

<211> 223

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

25

<223> Desmethylmenaquinone methyltransferase

<400> 170

<211> 672

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 171 <210> 173

<210> 172

<211> 223

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 172

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 173 5 <210> 174

<211> 223

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 174

<210> 175

<211> 672 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 175

<210> 176 15 <211> 223

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 176 <210> 177

<211> 675

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 177

<210> 178 15 <211> 224

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (21) ... (173) 25 <223> Desmethylmenaquinone methyltransferase

<400> 178

<210> 179

<211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 179

<210> 180 15 <211> 223

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 25 <223> Desmethylmenaquinone methyltransferase

<400> 180

<210> 181

<211> 687

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 181

<210> 182

<211> 228 15 <212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

<221> SIGNAL

<222> (1) ... (31)

25 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

30 <400> 182 <210> 183 <211> 693

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 183

10 15

<210> 184 <211> 230 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (21) ... (173) <223> Desmethylmenaquinone methyltransferase

<400> 184

<210> 185

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 185 5 <210> 186

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 186

<210> 187

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 187

<210> 188 15 <211> 225

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 188 <210> 189

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 189

<210> 190

<211> 225

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

25 <400> 190 <210> 191

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 191 5 <210> 192

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 192

<210> 193

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 193

<210> 194 15 <211> 225

<212> PRT

<213> Unknown

<220> 5 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 10 <223> Desmethylmenaquinone methyltransferase

<400> 194

<210> 195

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 195

<210> 196 15 <211> 225

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 25 <223> Desmethylmenaquinone methyltransferase

<400> 196 <212> DNA

<213> Unknown

<220> 10 <223> Obtained from an environmental sample

<400> 197 5 <210> 198

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 20 <222> (197) ... (200)

<223> N-glycosylation site. Prosite id = PS00001

<400> 198

<210> 199

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 199 5 <210> 200

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 20 <222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

<400> 200

<210> 201

<211> 633 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 201

<210> 202 15 <211> 210

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample <220>

<221> DOMAIN

<222> (6) ... (158)

<223> Desmethylmenaquinone methyltransferase

<400> 202

<210> 203 <211> 699

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 203

10 15

<210> 204 <211> 232 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (21) ... (173) <223> Desmethylmenaquinone methyltransferase

<400> 204

<210> 205

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 205

15 <210> 206

<211> 225

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (28) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 206

<210> 207

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 207

<210> 208 15 <211> 225

<212> PRT

<213> Unknown <220>

<223> Obtained from an environmental sample

5 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

10 <220>

<221> SITE

<222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

15 <400> 208 <210> 209

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 209

<210> 210 15 <211> 225

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 25 <223> Desmethylmenaquinone methyltransferase

<400> 210 <210> 211

<211> 702

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 212 10 <211> 233

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 20 <223> Desmethylmenaquinone methyltransferase

<400> 212

<210> 213

<211> 690 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 213 5 <210> 214

<211> 229

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 214

<210> 215

<211> 702 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 215

<210> 216 15 <211> 233

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (20) ... (172) 25 <223> Desmethylmenaquinone methyltransferase

<400> 216

<210> 217 5 <211> 702

<212> DNA

<213> Unknown

<220> 10 <223> Obtained from an environmental sample

<400> 217

15 <210> 218

<211> 233

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 218

<210> 219

<211> 672

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 219

15 <210> 220

<211> 223

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 220 <210> 221 <211> 672

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 221

10 15

<210> 222 <211> 223 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (20) ... (172) <223> Desmethylmenaquinone methyltransferase

<400> 222

<210> 223

<211> 678

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 225

<211> 672

<212>

 DNA 5 <213> Unknown

10 <400> 223

<210> 224

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 224

<220>

<223> Obtained from an environmental sample

10 <400> 225

15 <210> 226

<211> 223

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 226

<210> 227

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 227

<210> 228 15 <211> 225

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 25 <223> Desmethylmenaquinone methyltransferase <220>

<221> SITE

<222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

<400> 228 <210> 229

<211> 678

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 231

<211> 678

<212>

 DNA 5 <213> Unknown

10 <400> 229

<210> 230

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (151) ... (154)

30

<223> N-glycosylation site. Prosite id = PS00001

<400> 230

<220>

<223> Obtained from an environmental sample

10 <400> 231

15 <210> 232

<211> 225

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 232

<210> 233

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 233

<210> 234

<211> 225

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

fifteen

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (151) ... (154)

twenty

<223> N-glycosylation site. Prosite id = PS00001

<400> 234

<210> 235

<211> 678

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 237

<211> 678

<212>

 DNA 5 <213> Unknown

10 <400> 235

<210> 236

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 236

<220>

<223> Obtained from an environmental sample

10 <400> 237

<210> 238

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 238

<210> 239

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 239 5 <210> 240

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 20 <222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

<400> 240

<210> 241

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 241 <210> 242

<211> 225

<212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

10 <221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<220>

15 <221> SITE

<222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

<400> 242

<210> 243

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 243

<210> 244 15 <211> 225

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222>

 (22) ... (174) 25 <223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222>

 (151) ... (154) 30 <223> N-glycosylation site. Prosite id = PS00001

<400> 244

<210> 245

<211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 245

<210> 246

<211> 225

<212> PRT 15 <213> Unknown

<220>

<223> Obtained from an environmental sample

20 <220>

<221> DOMAIN

<222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

25 <220>

<221> SITE

<222> (2) ... (5)

<223> N-glycosylation site. Prosite id = PS00001

30 <400> 246 <212> DNA

<213> Unknown

<220> 10 <223> Obtained from an environmental sample

<400> 247 5 <210> 248

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 248

<210> 249

<211>

 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 249

<210> 251

<211>

 693 5 <212> DNA

<210> 250

<211> 225

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

fifteen

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (2) ... (5)

twenty

<223> N-glycosylation site. Prosite id = PS00001

<400> 250

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 251

<210> 252 15 <211> 230

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN <222> (27) ... (179)

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (156) ... (159)

<223> N-glycosylation site. Prosite id = PS00001

<400> 252

<210> 253

<211> 702

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 253

10 15

<210> 254 <211> 233 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (21) ... (173) <223> Desmethylmenaquinone methyltransferase

25

<220> <221> SITE <222> (229) ... (232) <223> N-glycosylation site. Prosite id = PS00001

<400> 254

5

<210> 255 <211> 681 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample

<400> 255

478

5 <210> 256

<211> 226

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 256

<210> 257

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 257 5 <210> 258

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 258

<210> 259

<211> 579 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 259

<210> 260 15 <211> 192

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (1) ... (132) 25 <223> Desmethylmenaquinone methyltransferase

<400> 260 5 <210> 261

<211> 678

<212> DNA

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<400> 261 5 <210> 262

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 262 <210> 263 <211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 263 <210> 265 <211> 678

10 15

<210> 264 <211> 225 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (22) ... (174) <223> Desmethylmenaquinone methyltransferase

<400> 264

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 265

10 15

<210> 266 <211> 225 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (22) ... (174) <223> Desmethylmenaquinone methyltransferase

<400> 266

5

<210> 267 <211> 678 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample <400> 267

491

5 <210> 268

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 268

<210> 269

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 269

<210> 270

<211> 225

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

fifteen

<223> Desmethylmenaquinone methyltransferase

<400> 270

<210> 271

<211> 678

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 271 <210> 273

<210> 272

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 272

<211>

 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 273

<210> 275

<211>

 690 5 <212> DNA

<210> 274

<211> 225

5

<212> PRT

<213> Unknown

<220>

10

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

fifteen

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (151) ... (154)

twenty

<223> N-glycosylation site. Prosite id = PS00001

<400> 274

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 275 5 <210> 276

<211> 229

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 276

<210> 277

<211> 678

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<400> 277

<210> 278

<211> 225

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

twenty

<223> Desmethylmenaquinone methyltransferase

<400> 278

<210> 279

<211> 678

<212>

 DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 281

<211> 678

<212>

 DNA 5 <213> Unknown

10 <400> 279

<210> 280

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<400> 280

<220>

<223> Obtained from an environmental sample

10 <400> 281

15 <210> 282

<211> 225

<212> PRT

<213> Unknown

20 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 25 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 282 <210> 283 <211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 283 <210> 285

10 15

<210> 284 <211> 225 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (22) ... (174) <223> Desmethylmenaquinone methyltransferase

<400> 284

<211> 678

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 285

<210> 286 10 <211> 225

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174) 20 <223> Desmethylmenaquinone methyltransferase

<400> 286 <210> 287 <211> 678

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 288

<211> 225

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (22) ... (174)

25

<223> Desmethylmenaquinone methyltransferase

<220>

<221> SITE

<222> (151) ... (154)

30

<223> N-glycosylation site. Prosite id = PS00001

<400> 288

<210> 289

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 289 5 <210> 290

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<220>

<221>

 SITE 20 <222> (151) ... (154)

<223> N-glycosylation site. Prosite id = PS00001

<400> 290

<210> 291

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 291

<210> 292

<211> 225

<212> PRT

<213> Unknown

5 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 10 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 292

5

<210> 293 <211> 678 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample 521

<400> 293

5 <210> 294

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 294 <210> 295

<211> 678 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 295 5 <210> 296

<211> 225

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (22) ... (174)

<223> Desmethylmenaquinone methyltransferase

<400> 296 <210> 297

<211> 672 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 297

10 <210> 298

<211> 223

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (20) ... (172)

<223> Desmethylmenaquinone methyltransferase

<400> 298 <210> 299

<211> 501

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 300

<211> 166

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (2) ... (159)

<223> Desmethylmenaquinone methyltransferase

20 <400> 300 <210> 301

<211> 501 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 301

<210> 302 15 <211> 166

<212> PRT

<213> Unknown

5 10

<220> <223> Obtained from an environmental sample <220> <221> DOMAIN <222> (2) ... (159) <223> Desmethylmenaquinone methyltransferase <400> 302

<210> 303

<211> 501

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<210> 304

<211> 166

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (2) ... (159)

25

<223> Desmethylmenaquinone methyltransferase

<400> 304

5 <210> 305

<211> 639

<212> DNA

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<400> 305 <210> 306

<211> 212 5 <212> PRT

<213> Unknown

<220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (1) ... (195)

<223> KDPG and KHG aldolase

<220>

<221> SITE

<222> (32) ... (41)

<223> Active site of KDPG and KHG aldolases. Prosite id = PS00159

<400> 306 <210> 307

<211> 639

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 307

10 15

<210> 308 <211> 212 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (2) ... (199) <223> KDPG and KHG aldolase

<400> 308

<210> 309

<211> 687

<212> DNA 5 <213> Unknown

<220>

<223> Obtained from an environmental sample

10 <400> 309

<210> 310

<211> 228

fifteen

<212> PRT

<213> Unknown

<220>

twenty

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (16) ... (215)

25

<223> KDPG and KHG aldolase

<400> 310

<210> 311

<211> 600 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 311

<210> 312 15 <211> 200

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (4) ... (198) 25 <223> KDPG and KHG aldolase

<400> 312

5

<210> 313 <211> 681 <212> DNA <213> Unknown

10

<220>

540

<223> Obtained from an environmental sample

<210> 314

<211> 226

10

<212> PRT

<213> Unknown

<220>

fifteen

<223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (12) ... (207)

twenty

<223> KDPG and KHG aldolase

<400> 314

<210> 315

<211> 576 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 315

<210> 316 15 <211> 192

<212> PRT

<213> Unknown

<220> 20 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (9) ... (192) 25 <223> KDPG and KHG aldolase <220>

<221> SITE

<222> (176) ... (179)

<223> N-glycosylation site. Prosite id = PS00001

<400> 316

10 <210> 317

<211> 612

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 317

10 <210> 318

<211> 203

<212> PRT

<213> Unknown

15 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 20 <222> (1) ... (192)

<223> KDPG and KHG aldolase

<400> 318 <210> 319

<211> 600

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 319

<210> 320 10 <211> 199

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222>

 (1) ... (186) 20 <223> KDPG and KHG aldolase

<220>

<221> SITE

<222>

 (23) ... (32) 25 <223> Active site of KDPG and KHG aldolases. Prosite id = PS00159

<220>

<221> SITE

<222>

 (112) ... (125) 30 <223> Residue that forms a Schiff base of KDPG and KHG aldolases. Prosite id = PS00160

<400> 320

5

<210> 321 <211> 654 <212> DNA <213> Unknown

10

<220> <223> Obtained from an environmental sample

548

<400> 321

5 <210> 322

<211> 217

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (9) ... (204)

<223> KDPG and KHG aldolase

<400> 322 <210> 323

<211> 603 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 323 5 <210> 324

<211> 201

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221>

 DOMAIN 15 <222> (9) ... (201)

<223> KDPG and KHG aldolase

<220>

<221>

 SITE 20 <222> (40) ... (49)

<223> Active site of KDPG and KHG aldolases. Prosite id = PS00159

<400> 324 <210> 325 <211> 648

<212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 325 <210> 327

10 15

<210> 326 <211> 215 <212> PRT <213> Unknown <220> <223> Obtained from an environmental sample

twenty

<220> <221> DOMAIN <222> (1) ... (200) <223> KDPG and KHG aldolase

25

<220> <221> SITE <222> (104) ... (107) <223> N-glycosylation site. Prosite id = PS00001

<400> 326

<211> 642

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 328

<211> 213

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (5) ... (200)

<223> KDPG and KHG aldolase

20 <220>

<221> SITE

<222> (37) ... (96)

<223> Active site of KDPG and KHG aldolases. Prosite id = PS00159

25 <400> 328 <210> 329

<211> 618 5 <212> DNA

<213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 329 5 <210> 330

<211> 205

<212> PRT

<213> Unknown

10 <220>

<223> Obtained from an environmental sample

<220>

<221> DOMAIN 15 <222> (4) ... (199)

<223> KDPG and KHG aldolase

<400> 330 <210> 331

<211> 663

<212> DNA <213> Unknown

<220>

<223> Obtained from an environmental sample

<400> 331

<210> 332 10 <211> 220

<212> PRT

<213> Unknown

<220> 15 <223> Obtained from an environmental sample

<220>

<221> DOMAIN

<222> (15) ... (211) 20 <223> KDPG and KHG aldolase

<400> 332 <210> 333

<211> 648

<212> DNA

<213> Unknown <220>

<223> Obtained from an environmental sample

<210> 334

<211> 215

<212> PRT 10 <213> Unknown

<220>

<223> Obtained from an environmental sample

15 <220>

<221> DOMAIN

<222> (12) ... (206)

<223> KDPG and KHG aldolase

20 <220>

<221> SITE

<222> (44) ... (53)

<223> Active site of KDPG and KHG aldolases. Prosite id = PS00159

25 <220>

<221> SITE

<222> (133) ... (146)

<223> Residue that forms a Schiff base of KDPG and KHG aldolases. Prosite id = PS00160

30 <400> 334 <210> 335

<211> 19

<212> DNA <213> Artificial Sequence

<220> <223> Primer

<220> <221> miscellaneous characteristic <222> (1) .. (19) <223> b is c, g or t

<220> <221> miscellaneous characteristic <222> (1) .. (19) <223> and is c or t

<220> <221> miscellaneous characteristic <222> (1) .. (19) <223> r is a or g

<220> <221> miscellaneous characteristic <222> (1) .. (19) <223> w is a or t

<400> 335 aargtbtwyg argacaatg

19

<210> 336 <211> 18 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<220> <221> miscellaneous characteristic <222> (1) .. (18) <223> d is a, g or t

<220> <221> miscellaneous characteristic <222> (1) .. (18) <223> and is c or t

<220> <221> miscellaneous characteristic <222> (1) .. (18) <223> r is a or g

<220> <221> miscellaneous characteristic <222> (1) .. (18) <223> w is a or t

<400> 336 gcdcagawca ayggrtgg

18

<210> 337 <211> 23 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<220> <221> miscellaneous characteristic <222> (1) .. (23) <223> d is a, g or t

<220> <221> miscellaneous characteristic <222> (1) .. (23) <223> and is c or t

<220> <221> miscellaneous characteristic <222> (1) .. (23) <223> r is a or g

<220> <221> miscellaneous characteristic <222> (1) .. (23) <223> s is c or g

<400> 337 ccatcrsyat cdgcrtadag cca

2. 3

<210> 338 <211> 20 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<220> <221> miscellaneous characteristic <222> (1) .. (20) <223> d is a, g or t

<220> <221> miscellaneous characteristic <222> (1) .. (20) <223> and is c or t

<220> <221> miscellaneous characteristic <222> (1) .. (20) <223> r is a or g

<220> <221> miscellaneous characteristic <222> (1) .. (20) <223> n is a, c, g or t

<400> 338 gcrtadagcc aytcnccrtc

 twenty

<210> 339 <211> 30 <212> DNA <213> Artificial Sequence

<220>

<223> primer

<400> 339 ataagacata tgcctatcgt tgttacgaag

30

<210> 340 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 340 ataagaggat ccttattcct cgggcagccg etc

33

<210> 341 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 341 ataagacata tgaacagacc tgtggttgtc

30

<210> 342 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 342 ataagaggat ccttacaggt acttgagacc gag

33

<210> 343 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 343 ataagacata tgagcgtggt catccgaaac

30

<210> 344 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 344 ataagaggat ccttacttcg ctttgttata ggc

33

<210> 345 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 345 ataagacata tgaacaagcc cgtggttgtg

30

<210> 346 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 346 ataagaggat ccttacaagt acttgagacc gagg

3. 4

<210> 347 <211> 29 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 347 ataagacata tgagcgtggt cgtcaccgg

29

<210> 348 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 348 ataagaggat ccttagccgt ttttcccgtc ggtg

3. 4

<210> 349 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 349 agaagacata tgatgagcat cgtcgtccag aac

33

<210> 350 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 350 agaagaggat cctcagacat atttcaggcc cttg

3. 4

<210> 351 <211> 30

<212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 351 agaagacata tgatgagcgt ggtcatcacc

30

<210> 352 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 352 acaacaggat ccctatttct tctccggcgt ttc

33

<210> 353 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 353 ataatacata tgagcgtcgt cgttcagaac

30

<210> 354 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 354 ataataggat ccttagacat atttgagccc cttc

3. 4

<210> 355 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 355 agaagacata tgatgtcggt tgtcgttcag aac

33

<210> 356 <211> 31 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 356 agaagaggat cctcagatat acttcaggcc c

31

<210> 357

<211> 852

<212> DNA

<213> Artificial Sequence

<220>

<223> ATCC 4978 DAAT

<210> 358

<211> 283

<212> PRT 15 <213> Artificial Sequence

<220>

<223> ATCC 4978 DAAT

20 <400> 358

<210> 359 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 359 gtgattgttt catcaacgaa ttcagaagta acgcc

35

<210> 360 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 360 gtgattgttt catcaacgcg ttcagaagta acgcc

35

<210> 361 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 361 gtgattgttt catcaacgag ttcagaagta acgcc

35

<210> 362 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 362 gtgattgttt catcaacggc ttcagaagta acgcc

35

<210> 363 <211> 31 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 363 gtgcaggccc tcgtgctcat attttccctg g

31

<210> 364 <211> 45 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 364 gaagtgattg tttcatcaac gcagtcagaa gtaacgccaa ttatc

Four. Five

<210> 365 <211> 40 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 365 catatgagtt atagcttatg gaatgaccaa attgtgaatg

40

<210> 366 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 366 ctcgagtgcg gccgcaagct tgtcgacgga gctc

3. 4

<210> 367 <211> 95 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 367

<210> 368 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 368 ggccagtgaa ttgtaatacg actcactata gggc

3. 4

<210> 369 <211> 42 <212> DNA <213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 369 cgccatcttt aattccataa atatttgaag aagagccttc tg

42

<210> 370 <211> 66 <212> DNA <213> Artificial Sequence

<220>

<223> Mutagenic Primer

<400> 370

<210> 371

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> Mutagenic Primer

<400> 371 gtattataca cacatccagc gaataactac atcttaaatg gtattacacg tcaag 55

<210> 372

<211> 64

<212> DNA

<213> Artificial Sequence

<220>

<223> Mutagenic Primer

<400> 372

<210> 373

<211> 64

<212> DNA

<213> Artificial Sequence

<220>

<223> Mutagenic Primer

<400> 373

<210> 374

<211> 64

<212> DNA

<213> Artificial Sequence

<220>

<223> Mutagenic Primer

<400> 374

<210> 375

<211> 16

<212> PRT

<213> P. striata

<220>

<221> miscellaneous feature

<222> (12) .. (12)

<223> Xaa can be any natural amino acid

<400> 375 <210> 379

<210> 376 <211> 28 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 376 agaagacata tgccctttcg ccgtaggg

28

<210> 377 <211> 32 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 377 agaagaggat cctcagtcga cgagtatctt cg

32

<210> 378 <211> 1230 <212> DNA <213> Artificial Sequence

<220> <223> KT2440 BAR

<400> 378

<211> 409 5 <212> PRT

<213> Artificial Sequence

<220>

<223> KT2440 BAR

<400> 379

<210> 380

<211> 1152

<212> DNA

<213> Geobacillus stearothermophilus

<400> 380

5 <210> 381

<211> 384

<212> PRT

<213> Geobacillus stearothermophilus

10 <400> 381

<210> 382

<211> 43

<212> DNA

<213> Artificial Sequence

<220> <223> Mutagenic primer

<400> 382 gccatttgga aacgatcaac gcggaagtgc cttgcacgat cag

43

<210> 383 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 383 gatataccat ggcatactca ttatggaatg

30

<210> 384 <211> 32 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 384 gttatcggat ccttaggcat taattgaaat tg

32

<210> 385 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 385 gaggagctcg agtcagacgt atttcagtcc tttttc

36

<210> 386 <211> 29 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 386 agaagacata tgatttatca gccggggac

29

<210> 387 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 387 agaagacata tgggtgtcgt cgtccaaaac

30

<210> 388 <211> 31

<212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 388 ataataggat ccttagacat atttgaggcc c

31

<210> 389 <211> 28 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 389 ataatacata tgaagccggt ggtggtgc

 28

<210> 390 <211> 31 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 390 agaagaggat ccttagacat aggtgagccc c

31

<210> 391 <211> 26 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 391 ataataccat gggtgtcgtg gtccag

26

<210> 392 <211> 32 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 392 agaagaggat ccttagacat atttcaggcc cc

32

<210> 393 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 393 gcggaacata tgtttgagaa cattaccgcc

30

<210> 394

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 394 ataaccggat ccttacagca ctgccacaat cg 32

<210> 395

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 395 cgctcttatg gttcggtttg cttgggttgc tcaccc 36

<210> 396

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 396 gggtgagcaa cccaagcttt ccgaaccata agagcg 36

<210> 397

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 397 caaaaaatac cagcgttaag ggagtgtggg tgagcaacc 39

<210> 398

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 398 cattaccgcc gctactgccg acccgattc 29

<210> 399

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 399

caccaaaaat tacctcggcg tagacggcat ccctgaatt

39

<210> 400 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 400 tgatgcggaa aatcacgctc ttgacttcga tgcac

35

<210> 401 <211> 35 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 401 gcggcgccat ggaaaatgat ccgattggtc taatg

35

<210> 402 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 402 gcggcggtcg acgcaattac aattgtgttt gtc

33

<210> 403 <211> 34 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 403 gcggcgccat ggatgtatgt ataattttat ttag

3. 4

<210> 404 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 404 gcggcggtcg acaaatttca ttattcattc taattt

36

<210> 405 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Primer

<400> 405 ggccggcata tgtcgatcct taacgactac aaacgt

36

5

<210> 406 <211> 36 <212> DNA <213> Artificial Sequence

<220> <223> Primer

fifteen

<400> 406 ggaaggctcg agtcatgatt ggtttccaga caaatt <210> 407 <211> 27 <212> PRT <213> Artificial Sequence 36

<220> <223> Synthetic Signal Sequence

<210> 408 <211> 28 <212> DNA <213> Artificial Sequence

<220> <223> Primer

35

<400> 408 ataatacata tgcccttctc ccgtaccc  28

<210> 409 <211> 31 <212> DNA <213> Artificial Sequence

Four. Five

<220> <223> Primer <400> 409 gcggcgggat ccttactgat ctttcaggat t 31

<210> 410 <211> 1230 <212> DNA <213> Artificial Sequence <223> Arginine racemase from Pseudomonas taetrolens

55

<400> 410

5

<210> 411 <211> 29 <212> DNA <213> Artificial Sequence

10

<220> <223> Primer

15 20

<400> 411 tacccaggct gagatggaag acatcaacg <210> 412 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer 29

<400> 412

586

gccagcaacg argargcmcg cgt

2. 3

5

<210> 413 <211> 18 <212> DNA <213> Artificial Sequence

10

<220> <223> Primer <400> 413 tggccstkga tcagcaca  18

fifteen

<210> 414 <211> 716 <212> DNA <213> Artificial Sequence

twenty

<220> <223> PCR product of A. caviae

<210> 415

<211> 238

<212> PRT 30 <213> Aeromonas caviae

<220>

<221> MISCELLANEOUS FEATURE

<222> (237) .. (237) 35 <223> Xaa can be Phe, Gln, Asn or Lys

<400> 415

<210> 416

<211> 1227

<212> DNA

<213> Aeromonas caviae

<400> 416

5 <210> 417

<211> 408

<212> PRT

<213> Aeromonas caviae

10 <400> 417

Claims (16)

1.- A method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, the derivatives being of (R, R) -monatin compounds of general formula (II): wherein Ra, Rb, Rc, Rd and Re each independently represent any substituent that is selected from a hydrogen atom, a hydroxyl group, a C1-C3 alkyl group, a C1-C3 alkoxy group, an amino group or an atom of halogen, which is selected from an iodine atom, bromine atom, chlorine atom or fluorine atom, provided that Ra, Rb, Rc, Rd, and Re are not all hydrogen and where Rb and Rc, and / or Rd and Re together form a C1-C4 alkylene group, where a pathway reaction is facilitated by a polypeptide that is selected from an isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 276 or fragments or subsequences thereof that have aldolase activity. 2. The method of claim 1, wherein a multi-stage pathway reaction is further facilitated by one or more polypeptides that are selected from isolated or recombinant polypeptides comprising the amino acid sequence of SEQ ID NO: 2 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO : 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 , SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO : 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 106 , SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ I D NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO : 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 278, SEQ ID NO: 282, SEQ ID NO: 284 , SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO : 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof that have aldolase activity. 3. The method of claim 1, wherein one or more stages in said multi-stage pathway is a chemical stage. 4. The method of claim 1, wherein the reaction is a reaction between indole-3-pyruvate and a C3 carbon source. 5. The method of claim 4, wherein the polypeptide (s) are selected from polypeptides comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 28 , SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 106, SEQ ID NO : 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 148 , SEQ ID NO: 156, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 192, SEQ ID NO: 200, SEQ ID NO: 218, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 236, SEQ ID NO : 238, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272 , SEQ ID NO: 278, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 298, SEQ ID NO: 300 or SEQ ID NO: 304 6. The method of claim 4, wherein the reaction produces R-2-hydroxy-2- (indole-3-yl-methyl) -4-ketoglutaric acid instead of S-2-hydroxy-2- (indole- 3-yl-methyl) -4-ketoglutaric. 7. The method of claim 6, wherein the polypeptide (s) are selected from polypeptides comprising the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 116, SEQ ID NO: 298, SEQ ID NO: 44 , SEQ ID NO: 54, SEQ ID NO: 148, SEQ ID NO: 46, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 122, SEQ ID NO: 74, SEQ ID NO: 64, SEQ ID NO: 108, SEQ ID NO: 96, SEQ ID NO: 126, SEQ ID NO: 80, SEQ ID NO: 36, SEQ ID NO: 62, SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO : 94, SEQ ID NO: 58, SEQ ID NO: 50, SEQ ID NO: 106, SEQ ID NO: 42, SEQ ID NO: 278, SEQ ID NO: 162, SEQ ID NO: 178, SEQ ID NO: 166 , SEQ ID NO: 218, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 244, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 264, SEQ ID NO: 268, SEQ ID NO: 272, SEQ ID NO: 184, SEQ ID NO: 282, SEQ ID NO: 186, SEQ ID NO: 192, SEQ ID NO: 200, SEQ ID NO: 284, SEQ ID NO: 172, SEQ ID NO : 180, SEQ ID NO: 168, SEQ ID NO: 228, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240 or SEQ ID NO: 156. 8. The method of claim 1, wherein the product is selected from (R, R) -monatin, salts thereof, and combinations thereof. 9. The method of claim 1, wherein the reaction facilitated by said polypeptide or polypeptides is carried out in 1.0 to 10.0 mM MgCl2 or pH 7.0 to pH 11.5 or 0.005% at 1% polysorbate detergent. 10. The method of claim 7, wherein (R, R) -monatin is produced in larger amounts than (S, S) monatin, (S, R) -monatin or (R, S) -monatin. 11. The method of claim 1, wherein the fragments or subsequences thereof have an aldolase activity of at least 0.2 mg MP / mg protein / h. 12.- A method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, the derivatives being of (R, R) monatin compounds of general formula (II): wherein Ra, Rb, Rc, Rd and Re each independently represent any substituent that is selected from a hydrogen atom, a hydroxyl group, a C1-C3 alkyl group, a C1-C3 alkoxy group, an amino group or an atom of halogen, which is selected from an iodine atom, bromine atom, chlorine atom or fluorine atom, provided that Ra, Rb, Rc, Rd, and Re are not all hydrogen and where Rb and Rc, and / or Rd and Re together form a C1-C4 alkylene group, where a pathway reaction is facilitated by a nucleic acid sequence encoded polypeptide comprising a sequence having a percent sequence identity of at least 95% relative to SEQ ID NO: 275. 13. The method of claim 12, wherein a multi-stage reaction is further facilitated by at least one polypeptide encoded by a nucleic acid sequence comprising a sequence having a percent sequence identity of at least 95% or more preferably 100% with respect to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, I KNOW THAT ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO : 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133 , SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO : 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183 , SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO : 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235 , SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO: 329, SEQ ID NO: 331, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337 or SEQ ID NO: 338. 14.- A method comprising: producing a chosen product of (R, R) -monatin, derivatives of (R, R) -monatin, salts thereof and combinations thereof in a multi-stage route, the derivatives being of (R, R) monatin compounds of general formula (II): wherein Ra, Rb, Rc, Rd and Re each independently represent any substituent that is selected from a hydrogen atom, a hydroxyl group, a C1-C3 alkyl group, a C1-C3 alkoxy group, an amino group or an atom of halogen, which is selected from an iodine atom, bromine atom, chlorine atom or fluorine atom, provided that Ra, Rb, Rc, Rd, and Re are not all hydrogen and where Rb and Rc, and / or Rd and Re together form a C1-C4 alkylene group, where a pathway reaction is facilitated by a nucleic acid sequence encoded polypeptide that has sufficient sequence homology to remain hybridized under stringent conditions to a nucleic acid of SEQ ID NO: 275, where stringent conditions are no less stringent than wash with 0.15M NaCl for 15 minutes at 72 ° C. 15.- A method comprising: producing a chosen product of R-2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid, salts thereof and combinations thereof in one way, wherein a reaction in the pathway is facilitated by a polypeptide chosen from isolated or recombinant polypeptides comprising the amino acid sequence of SEQ ID NO: 276 or fragments or subsequences thereof having aldolase activity, wherein said R-2-hydroxy acid 2- (indole-3 -ylmethyl) -4-ketoglutaric, salts thereof and combinations thereof are used to prepare a sweetening product. 16. The method of claim 15, wherein a reaction in the pathway is further facilitated by one or more polypeptides that are selected from isolated or recombinant polypeptides comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 , SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO : 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164 , SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO : 198, SEQ ID NO: 200, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216 , SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID N O: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 268, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 278, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, SEQ ID NO: 288, SEQ ID NO: 290, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300, SEQ ID NO: 302, SEQ ID NO: 304, SEQ ID NO: 306, SEQ ID NO: 308, SEQ ID NO: 310, SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326, SEQ ID NO: 328, SEQ ID NO: 330, SEQ ID NO: 332 or SEQ ID NO: 334 or fragments or subsequences thereof having aldolase activity, wherein said R-2-hydroxy 2- (indole-3-ylmethyl) -4-ketoglutaric acid, salts thereof and combinations thereof is used to prepare a sweetening product. FIG. fifteen